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Most bacteria are surrounded by a peptidoglycan cell wall that defines their shape and protects them from osmotic lysis. The expansion and division of this structure therefore plays an integral role in bacterial growth and division. Additionally, the biogenesis of the peptidoglycan layer is the target of many of our most effective antibiotics. Thus, a better understanding of how the cell wall is built will enable the development of new therapies to combat the rise of drug-resistant bacterial infections. This review covers recent advances in defining the mechanisms involved in assembling the peptidoglycan layer with an emphasis on discoveries related to the function and regulation of the cell elongation and division machineries in the model organisms Escherichia coli and Bacillus subtilis.

Background The combination aztreonam/avibactam is currently under Phase 3 trials for the treatment of serious infections caused by Gram-negative bacteria including those with MBLs. Objectives To investigate the resistance mechanisms in Enterobacterales exhibiting aztreonam/avibactam MICs of ≥4 mg/L. Methods Among 8787 Enterobacterales, 17 (0.2%) isolates exhibited an aztreonam/avibactam MIC of ≥4 mg/L. Isolates were sequenced and screened for β-lactamases. Sequences of porins, penicillin-binding protein 3 (PBP3) and expression levels of AmpC and AcrA were evaluated. Results Eleven (11/4154 isolates; 0.26%) Escherichia coli, three (3/1981; 0.15%) Klebsiella pneumoniae and three (3/628; 0.5%) Enterobacter cloacae were identified. All E. coli showed either an ‘YRIK’ or ‘YRIN’ insertion in PBP3. In general, these isolates carried blaCMY and/or blaCTX-M variants, except for one isolate from Korea that also produced NDM-5 and one isolate from Turkey that produced OXA-48. Two DHA-1-producing K. pneumoniae overexpressed acrA and had a premature stop codon in either OmpK35 or OmpK36, whereas a third K. pneumoniae carried blaPER-2 and had a premature stop codon in OmpK35. All three E. cloacae expressed AmpC at levels ≥570-fold, but sequence analysis did not reveal known amino acid alterations associated with decreased avibactam binding or increased hydrolysis of β-lactams. Minor amino acid polymorphisms within OmpC, OmpF and PBP3 were noted among the E. cloacae. Conclusions A small number of isolates (0.2%) met the inclusion criteria. E. coli showed altered PBP3 as the most relevant resistance mechanism, whereas K. pneumoniae had multiple resistance mechanisms. Further investigations are needed to clarify resistance in E. cloacae.

Previous research has shown that penicillin-binding proteins (PBPs), enzymes involved in peptidoglycan (PG) assembly, could play an important role during the induction of the viable but nonculturable (VBNC) state, which allows non-spore-forming bacteria to survive adverse environmental conditions. The current study found that Clavibacter michiganensis has seven PBPs. Mutant analysis indicated that deletion of either of the class B PBPs was lethal and that the class A PBPs had an important role in PG synthesis, with the ΔpbpC mutant having an altered cellular morphology that resulted in longer cells that were swollen at one end and had thinner cell walls. The ΔpbpC mutant was also found to produce mucoid colonies in solid culture and a lower final cell titer in liquid medium, as well as having high sensitivity to osmotic stress and lysozyme treatment and surprisingly high pathogenicity. The double mutant, ΔdacB/ΔpbpE, also had a slightly altered phenotype, resulting in longer cells. Further analysis revealed that both mutants had high sensitivity to copper, which resulted in quicker induction into the VBNC state. However, only the ΔpbpC mutant had significantly reduced survivorship in the VBNC state. The study also confirmed that the VBNC state significantly improved the survivorship of wild-type C. michiganensis cells in response to environmental stresses and systemically demonstrated the protective role of the VBNC state in C. michiganensis, which is an important finding regarding its epidemiology and has serious implications for disease management.

Streptococcus pneumoniae is an important causal agent of pneumonia, meningitis, sepsis, bacteremia, and otitis media. Penicillin resistance rates in S. pneumoniae have remained stable in Argentina in the last years. In the late '90s more isolates with MIC of penicillin ≥2μg/ml were observed; however, their frequency has decreased in recent years. The phenotypic expression of penicillin resistance is due to a modification in penicillin-binding proteins associated with a mosaic structure in the coding genes. The expansion of successful resistant clones varies among the different regions and is influenced by the use of antibiotics, vaccines, particularly conjugated ones, as well as population density. Parenteral treatment with high doses of penicillin G continues to be effective for the treatment of pneumonia and bacteremia, oral aminopenicillins for otitis media and sinusitis and third generation cephalosporins for meningitis.

Ida Ayu Evangelina,1 Yetty Herdiyati,2 Avi Laviana,1 Rasmi Rikmasari,3 Cucu Zubaedah,4 Anisah,5 Dikdik Kurnia5 1Department of Orthodontics, Faculty of Dentistry – Universitas Padjadjaran, Bandung, Indonesia; 2Department of Pediatric Dentistry, Faculty of Dentistry – Universitas Padjadjaran, Bandung, Indonesia; 3Department of Prosthodontics, Faculty of Dentistry – Universitas Padjadjaran, Bandung, Indonesia; 4Department of Dental Public Health, Faculty of Dentistry – Universitas Padjadjaran, Bandung, Indonesia; 5Department of Chemistry, Faculty of Mathematics and Natural Science – Universitas Padjadjaran, Sumedang, IndonesiaCorrespondence: Dikdik KurniaDepartment of Chemistry, Faculty of Mathematics and Natural Science – Universitas Padjadjaran, Jl. Raya Bandung Sumedang km.21, Hegarmanah, Jatinangor, Kabupaten, Sumedang, Jawa Barat, 45363, IndonesiaTel/Fax +62 22 7794391Email dikdik.kurnia@unpad.ac.idBackground: Dental caries is a widespread disease that causes dental tissue destruction and leads to local and general complications. Gram-positive bacteria including Streptococcus mutans, Streptococcus sanguinis, and Enterococcus faecalis take part in dental caries formation. Gram-positive bacteria have cell walls that consistof a thick layer of peptidoglycan which maintains the strength and rigidity of the bacteria, as well as bacteria guard from internal osmotic pressure. The biosynthesis of peptidoglycan involves many enzymes, including the Mur family, penicillin binding protein (PBP), and sortases.Purpose: This research has the intention to screen and examine the antibacterial compound of edible plant Kemangi (Ocimum basilicum L.) in terms of how it fights against some oral pathogenic bacteria of E. faecalis ATCC 29212, S. mutans ATCC 25175, and S. sanguinis ATCC 10566.Materials and Methods: The O. basilicum L. was macerated by several organic solvents to obtain the extracts, before then being purified using several combinations of chromatography methods and the compound was discovered via spectroscopic methods. For the assay against bacteria, the extracts and compounds were tested using agar well diffusion and microdilution assay.Results: The isolated compound was identified as β-sitosterol. The compound activity against bacteria was evaluated by in vitro assay against S. sanguinis ATCC 10566 and E. faecalis ATCC 29212 with the MIC and MBC value of 25,000 and 50,000 ppm, respectively. The compound was also tested by in silico study using the molecular docking method. The molecular interaction between β-sitosterol and the protein target showed a lower binding affinity value than the native ligand and other positive controls for each protein. Based on the amino acid residue bound to the ligands, β-sitosterol on MurA and SrtA is not competitive to the positive control, showing potential as a natural antibacterial agent. Meanwhile, on the MurB and PBP, β-sitosterol and positive control do compete with each other.Conclusion: The compound, isolated from O. basilicum L. leaf, was determined as β-sitosterol, which has the molecular formula C29H50O. The antibacterial activity of β-sitosterol by in vitro assay showed weak antibacterial activity, yet exhibited the potential to inhibit the biosynthesis of peptidoglycan and prevent bacteria cell wall formation by inhibiting MurA and SrtA activity via docking simulation.Keywords: β-sitosterol, Ocimum basilicum L., MurA enzyme, peptidoglycan, cell wall biosynthesis

Bakterien haben ein vielseitiges Erscheinungsbild, welches größtenteils durch die Zellwand und andere formgebende Komponenten beeinflusst wird. Trotz jahrzehntelanger Forschung auf dem Gebiet der Zellwandsynthese gibt es immer noch viele offene und ungeklärte Fragen zur Organisation des bakteriellen Peptidoglykan Sacculus, aber auch zur räumlichen und zeitlichen Verteilung der an der Peptidoglykan Synthese beteiligten Proteinen, die sogenannten Penicillin Binde Proteine oder kurz PBPs, welche die letzten Reaktionen der Peptidoglykansynthese katalysieren. Das Bacillus subtilis Genom kodiert für eine große Anzahl von verschiedenen PBPs mit verschiedenen enzymatischen Eigenschaften bei denen jedoch nicht immer bekannt ist, wann oder unter welchen Bedingungen diese aktiviert werden. Einige PBPs sind dafür bekannt mit Proteinkomplexen, Elongasom and Divisom genannt, zu interagieren, welche für die Zellwandsynthese zuständig sind und meiste durch Zytoskelettelemente wie MreB und FtsZ organisiert werden. Um die Lokalisation und Funktion besser zu verstehen, wurden in dieser Arbeit verschiedene PBPs und MreB, als Indikatoren für den Ort der Zellwandsynthese, unter verschiedenen Bedingungen untersucht, wobei der Fokus auf der zeitlichen und räumlichen Verteilung dieser Proteine lag. Fünf PBPs wurden exemplarisch aus den verschiedenen Klassen ausgewählt und n-Terminal mit einem mVenus-Fluorophor fusioniert. Diese wurden mit Hilfe einer hochauflösende zeitliche und räumliche Mikroskopie Technik die Einzel-Molekül Verfolgung („Single-molecule-tracking“ genannt) untersucht und der Diffusionskoeffizient der Moleküle wurde bestimmt. Zwei verschiedene dynamische Populationen wurden entdeckt und nachgewiesen: eine langsam diffundierende, wahrscheinlich enzymatisch aktive, und eine schnelle, frei bewegliche inaktive Molekül-Population. Wobei die meisten PBPs 50% langsame Moleküle aufweisen und damit ungefähr die Hälfte der Moleküle an Substrat gebunden ist. Darüber hinaus sollte die Reaktion der PBPs auf verschiedene Umweltfaktoren getestet werden, dazu wurden die Zellen mit osmotischen Stressoren (NaCl und Sorbitol) sowie mit Antibiotika (Vancomycin, Penicillin G, Nisin, Fosfomycin und Bacitracin) behandelt. Hierbei konnten Veränderungen des Diffusionskoeffizienten der Proteine festgestellt werden. Vor allem wenn die Verfügbarkeit der Peptidoglykanbausteine durch die Anwesenheit der Antibiotika abweicht. Auch MreB reagiert auf die verfügbare Menge von Peptidoglykanbausteinen mit einer Veränderung der dynamischen Populationen, allerdings nicht so stark wie die meisten untersuchten PBPs. Des Weiteren wurde an die Ergebnisse einer vorherigen Doktorarbeit der Arbeitsgruppe angeknüpft (geschrieben von Dr. Simon Dersch). Die Reaktionen von am Elongasom beteiligten Proteinen (PbpH, Pbp2a und MreB) und jeweiligen Deletionen (∆pbpA und ∆pbpH) bzw. Depletion (MreB wurde durch ein Cas9 System in der Zelle auf ein niedriges Niveau gebracht) wurden im genetischen Hintergrund mit denselben Antibiotika untersucht. Die Dynamiken der beiden redundanten Transpeptidasen PbpH und Pbp2a veränderten sich merkbar im korrespondierenden Deletionsstamm unter dem Einfluss der Antibiotika. Während das niedrige Niveau von MreB in der Zelle nur einen geringen Einfluss auf die Diffusionskonstanten von PbpH oder Pbp2a hat, verändert sich das Verhalten von PbpH doch stark unter dem Einfluss von Antibiotika., Bacteria have a highly varied appearance and shape, which is primarily affected by the cell wall and other components that give shape to the cells. Despite decades of research in the field of cell wall synthesis there are still many open questions regarding the organisation of the bacterial peptidoglycan sacculus, but also regarding the spatiotemporal distribution of proteins that are involved in peptidoglycan synthesis. An essential class of these, the so-called Penicillin Binding Proteins, or PBPs for short, are all performing the last steps and some additional steps of the peptidoglycan synthesis. The Bacillus subtilis genome codes for a large number of different PBPs, which have different enzymatic properties, and it is not yet clear for all of these proteins, when – or under which conditions – they become active. Some of the PBPs are known to interact with cell wall synthesis machineries, the so called elongasome or divisome, both being partly organised by cytoskeletal elements like MreB or FtsZ. To better understand their localisation and function, different PBPs and MreB were used in this thesis as indicators for the location of cell wall synthesis. They were investigated under different conditions, where the main focus was the spatiotemporal distribution of these proteins. Five different PBPs from different classes were selected, and n-terminally tagged with an mVenus fluorophore. These were then investigated with the help of a high-resolution microscopy method called single-molecule tracking, and the diffusion coefficients of the molecules were determined. Two different dynamic molecular populations could be discovered, one a slow-diffusing, likely enzymatically active population, and the other a fast-moving inactive population. Most PBPs demonstrate a 50% slow diffusing population, which means that roughly half of the molecules are bound to their substrate. Additionally, the reactions involving the PBPs were tested in the presence of different environmental conditions – to achieve this, the cells were treated with osmotic stressors (NaCl and sorbitol), as well as antibiotics (vancomycin, penicillin G, nisin, fosfomycin, bacitracin). Through these experiments, changes in the diffusion coefficients of the proteins were observed. Particularly the availability of peptidoglycan building blocks was altered by the presence of antibiotics. MreB also reacts to the available amounts of peptidoglycan building blocks through a change in its dynamic populations, but not as strongly as most studied PBPs. Additionally, this thesis builds on the results of a previous doctoral thesis from the same research group (written by Dr. Simon Dersch). Therefore, this thesis (written by Lisa Stuckenschneider) also investigated the elongasome components PbpH, Pbp2a and MreB through corresponding deletion (∆pbpA and ∆pbpH) and depletion genetic backgrounds (MreB levels were strongly reduced in the cell by a Cas9 system), with the same antibiotics. The dynamics of both of the redundant transpeptidases PbpH and Pbp2a change strongly in the corresponding deletion strains and under the influence of antibiotics. Although the decreased MreB levels in the cell had only a minor effect on the diffusion constants of PbpH or Pbp2a. The absence of MreB made the diffusion of PbpH more susceptible to antibiotic treatment, and to a similar degree as in the ∆pbpA background.

Bacterial cell division is a complex and highly regulated process requiring the coordination of many different proteins. Despite substantial work in model organisms, our understanding of the systems regulating cell division in non-canonical organisms, including critical human pathogens, is far from complete. One such organism is Staphylococcus aureus, a spherical bacterium that lacks known cell division regulatory proteins. Recent studies on GpsB, a protein conserved within the Firmicutes phylum, have provided insight into cell division regulation in S. aureus and other related organisms. It has been revealed that GpsB coordinates cell division and cell wall synthesis in multiple species by interacting with Penicillin Binding Proteins (PBPs) and other partners. In S. aureus, we have previously shown that GpsB directly regulates FtsZ polymerization. In this study, using Bacillus subtilis as a tool, we isolated intragenic and extragenic spontaneous suppressor mutants that abrogate the lethality of S. aureus GpsB overproduction in B. subtilis. Through characterization of these mutants, we identified several key residues important for the function of GpsB. Furthermore, we discovered an additional role for GpsB in wall teichoic acid (WTA) biosynthesis in S. aureus. Specifically, we show that GpsB directly interacts with the wall teichoic acid export protein TarG using a bacterial two-hybrid analysis. We also identified a three-residue motif in GpsB that is crucial for this interaction. Based on the analysis of the localization of TagG in B. subtilis and its homolog TarG in S. aureus, it appears that WTA machinery is a part of the divisome complex. As such, we show additional evidence to the growing body of work that suggests that along with peptidoglycan synthesis, WTA biosynthesis and export may take place at the site of cell division. Taken together, this research illustrates how GpsB performs an essential function in S. aureus by directly linking the tightly regulated cell cycle processes of cell division and WTA-mediated cell surface decoration.IMPORTANCE/AUTHOR SUMMARYCytokinesis in bacteria involves an intricate orchestration of several key cell division proteins and other factors involved in building a robust cell envelope. One of the key factors that differentiates Gram-positive bacteria from Gram-negative bacteria is the presence of teichoic acids interlaced within the Gram-positive cell wall. By characterizing the role of Staphylococcus aureus GpsB, an essential cell division protein in this organism, we have uncovered an additional role for GpsB in wall teichoic acids (WTA) biosynthesis. We show that GpsB directly interacts with TarG of the WTA export complex. We also show this function of GpsB may be conserved in other GpsB homologs as GpsB and the WTA exporter complex follow similar localization patterns. It has been suggested that WTA acts as a molecular signal to control the activity of autolytic enzymes, especially during the separation of conjoined daughter cells. Thus, our results reveal that GpsB, in addition to playing a role in cell division, may also help coordinate WTA biogenesis.

Three isatin derivatives, namely, 1-allyl-3-hydroxy-3-(6-oxocyclohex-1-en-1-yl)indolin-2-one, C17H17NO3, 1-ethyl-3-hydroxy-3-(6-oxocyclohex-1-en-1-yl)indolin-2-one, C16H17NO3, and 5-bromo-3-hydroxy-1-methyl-3-(6-oxocyclohex-1-en-1-yl)indolin-2-one, C15H14BrNO3, were synthesized, crystallized by the slow-evaporation technique, characterized by1H and13C NMR spectroscopy, and analysed by the single-crystal X-ray diffraction (XRD) method. Quantum chemical parameters, such as the energy of the highest occupied molecular orbital, energy of the lowest unoccupied molecular orbital, energy gap, electronic energy, ionization potential, chemical potential, global hardness, global softness and electrophilicity index, were calculated. The druglikeness and bioactivity scores of the compounds were calculated. The activities of these isatin derivatives against bacterial strains, such asEschericia coli,Proteus vulgaris,Shigella flexneri,Staphylococcus aureusandMicrococcus luteus, and the fungal strainAspergillus niger, were determined using the well-diffusion assay method. Molecular docking studies were carried out to predict the binding mode of the isatin compounds with the penicillin binding protein enzyme and to identify the interactions between the enzyme and the ligands under study.

Introduction Invasive meningococcal disease (IMD) caused by serogroup W meningococci belonging to the ST-11 complex (MenW:cc11) has been increasing globally since the early 2000s. Penicillin resistance among meningococci due to the production of beta-lactamase remains relatively rare. Isolates displaying resistance and reduced susceptibility to penicillin due to alterations in the penA gene (encoding Penicillin Binding Protein 2) are increasingly reported. In 2016, a penicillin-resistant clade of MenW:cc11 isolates with altered penA genes was identified in Australia. More recently, an increase in penicillin-resistant invasive MenW:cc11 isolates was observed in England. Here, we investigate the distribution of penicillin resistance among English invasive MenW:cc11 isolates. Methods Isolates from IMD cases in England from July 2010 to August 2019 underwent whole genome sequencing and antibiotic susceptibility testing as part of routine surveillance. The PubMLST Neisseria database was used to determine the distribution of penicillin resistance among English MenW:cc11 isolates and to identify other closely related isolates. Results Twenty-five out of 897 English invasive MenW:cc11 isolates were resistant to penicillin; identified among six distinct sublineages and a singleton. Expansion of the Australian penicillin-resistant clade included isolates from several new countries as well as 20 English isolates. A newly identified penicillin resistance-associated lineage was also identified among several countries. Conclusion Penicillin resistance among diverse MenW:cc11 isolates is increasing. Surveillance of antibiotic resistance among meningococci is essential to ensure continued effective use.

Objectives To investigate the spread of ceftriaxone-resistant Neisseria gonorrhoeae lineages similar to strains H041 (2009) and FC428 (2015), we characterized 55 strains collected in 2013 from hospitals across Japan. Methods Susceptibility testing and whole-genome sequencing. Results Susceptibility rates were 58% for cefixime and 98% for ceftriaxone. The 55 strains were whole-genome sequenced and classified into nine MLST-STs. MLST-ST1901 was the most prevalent (n = 19) followed by MLST-ST7363 (n = 12) and MLST-ST7359 (n = 11). The most prevalent penA [encoding penicillin binding protein 2 (PBP2)] mosaic types, based on the N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) scheme, were 10.001 (n = 20) followed by 34.001 (n = 13). The H041 and FC428 strains were not detected; however, a single ceftriaxone-resistant strain (TUM15748) with a MIC of 0.5 mg/L ceftriaxone was identified. The TUM15748 strain belonged to MLST-ST7359 and N. gonorrhoeae multiantigen sequence typing-ST6771, and had a novel PBP2 (PBP2TUM15748, penA type 169.001). The amino acid sequence of PBP2TUM15748 showed partial similarity to that of PBP2 from N. gonorrhoeae GU140106 and commensal Neisseria perflava and Neisseria cinerea. Natural transformation and recombination experiments using full-length TUM15748 penA showed that the ceftriaxone MICs of transformants increased 16-fold or more compared with the parental ceftriaxone-susceptible recipient strain (NG9807, belonging to MLST-ST7363). No ceftriaxone-resistant MLST-ST7359 strains have previously been reported. Conclusions We showed here that a ceftriaxone-susceptible lineage acquired a mutant PBP2 mosaic type, integrating partial PBP2 sequences from commensal Neisseria species, resulting in the emergence of ceftriaxone-resistant strains.

The emergence of multi drug resistant clone CC320 serotype19F/19A and their capsular (cps) antigenic variants due to selective pressures such as vaccine had been reported worldwide. Hence, it is important to identify the prevalent clones, sequence types and cps variants of serotype 19F/19A in India, where PCV13 has been recently introduced. Multi-locus sequence typing (MLST) was performed for all (n = 21) invasive S. pneumoniae isolates of serotype 19A (n = 5) and 19F (n = 16) collected between the years 2012 and 2018 from children less than 5 years. The genome characterization by whole genome sequencing for the Sequence types (STs) 320 and 271(n = 7) were performed and compared with another six Indian WGSs of similar STs available from the GPS platform. The predominant STs in the serotype 19F/19A study isolates were of CC320: ST 320, 236 and 271, associated with PMEN clone Taiwan19F-14. The WGSs of CC320 study isolates showed high genomic similarity to the Taiwan19F-14 clone, and the penicillin binding protein (PBP) amino acid sequence similarity was 100% for PBP1A, 93% for PBP 2B and 2X. Whilst PBP comparison with other global MDR ST320 strains revealed that the ST320 clones in India are of low-level penicillin resistance. The presence of a few ST320/19A/19F invasive isolates with high similarity to the Taiwan clone suggests slow and gradual expansion of Taiwan19F-14 associated CC320 clones in India. Since serotype 19F/19A is covered by PCV13 vaccine, the expansion of 19F/19A cones with non-PCV13 vaccine serotype in India should be monitored.

Penicillin-binding proteins are the primary targets for beta lactam drugs, which are main stay of treatment for Streptococcus pneumoniae. The emergence of increased penicillin resistance in meninge...

Non-aureus staphylococci are commonly found on dairy farms. Two rarely investigated species are Staphylococcus (S.) cohnii and S. urealyticus. Since multidrug-resistant S. cohnii and S. urealyticus are known, they may serve as an antimicrobial resistance (AMR) gene reservoir for harmful staphylococcal species. In our study, nine S. cohnii and six S. urealyticus isolates from German dairy farms were analyzed by whole-genome sequencing and AMR testing. The isolates harbored various AMR genes (aadD1, str, mecA, dfrC/K, tetK/L, ermC, lnuA, fexA, fusF, fosB6, qacG/H) and exhibited non-wildtype phenotypes (resistances) against chloramphenicol, clindamycin, erythromycin, fusidic acid, rifampicin, streptomycin, tetracycline, tiamulin and trimethoprim. Although 14/15 isolates lacked the blaZ, mecA and mecC genes, they showed reduced susceptibility to a number of beta-lactam antibiotics including cefoxitin (MIC 4–8 mg/L) and penicillin (MIC 0.25–0.5 mg/L). The specificity of cefoxitin susceptibility testing for mecA or mecC gene prediction in S. cohnii and S. urealyticus seems to be low. A comparison with penicillin-binding protein (PBP) amino acid sequences of S. aureus showed identities of only 70–80% with regard to PBP1, PBP2 and PBP3. In conclusion, S. cohnii and S. urealyticus from selected German dairy farms show multiple resistances to antimicrobial substances and may carry unknown antimicrobial resistance determinants.

The design and synthesis of narrow-spectrum antibiotics that target a specific bacterial strain, species, or group of species is a promising strategy for treating bacterial infections when the causative agent is known. In this work, we report the synthesis and evaluation of four new siderophore-β-lactam conjugates where the broad-spectrum β-lactam antibiotics cephalexin (Lex) and meropenem (Mem) are covalently attached to either enterobactin (Ent) or diglucosylated Ent (DGE) via a stable polyethylene glycol (PEG3) linker. These siderophore-β-lactam conjugates showed enhanced minimum inhibitory concentrations against Escherichia coli compared to the parent antibiotics. Uptake studies with uropathogenic E. coli CFT073 demonstrated that the DGE-β-lactams target the pathogen-associated catecholate siderophore receptor IroN. A comparative analysis of siderophore-β-lactams harboring ampicillin (Amp), Lex and Mem indicated that the DGE-Mem conjugate is advantageous because it targets IroN and exhibits low minimum inhibitory concentrations, fast time-kill kinetics, and enhanced stability to serine β-lactamases. Phase-contrast and fluorescence imaging of E. coli treated with the siderophore-β-lactam conjugates revealed cellular morphologies consistent with the inhibition of penicillin-binding proteins PBP3 (Ent/DGE-Amp/Lex) and PBP2 (Ent/DGE-Mem). Overall, this work illuminates the uptake and cell-killing activity of Ent- and DGE-β-lactam conjugates against E. coli and supports that native siderophore scaffolds provide the opportunity for narrowing the activity spectrum of antibiotics in clinical use and targeting pathogenicity., Siderophore-β-lactam conjugates based on enterobactin and diglucosylated enterobactin enter the periplasm of uropathogenic E. coli CFT073 via the FepA and IroN transporters, and target penicillin-binding proteins.

Background: Methicillin-Resistant Staphylococcus aureus (MRSA) has become a major pathogene and its incidence continues to increase in various countries. The resistance to methicillin is caused by the mecA gene that encodes the expression of Penicillin Binding Protein 2a (PBP2a) so it has a low affinity for beta-lactam antibiotics. One of the factors that can increase the incidence of MRSA is inappropriate antibiotics use. Objective: This study is to identify presence of the mecA gene in MRSA and to compare histories of inappropriate antibiotic uses of healthcare workers and communities in Banyumas. Methods: This was a cross-sectional study conducting 120 samples from nasal swabs. Isolates of MRSA tested by bacteriological examinations and PCR of the mecA genes. Histories of antibiotic uses were assessed by using questionnaires and then analysed descriptively by using Fisher Exact test (SPPS, version 20). Result: One sample of S. aureus from the microbiology examination (0.83%) was an MRSA although there was no a mecA gene identified by using the PCR method. 66.1% of the healthcare workers and 88,3% of the communities used antibiotics inappropriately. The results showed that there was a statistically significant difference in the histories of irrational uses of antibiotics (p?0.05). Conclusion: A number of the communities who used inappropriate antibiotics was higher than the health workers. Furthermore, one MRSA strain with a negative mecA gene was found in the health worker group.

Antibiotic resistance has become a major world health challenge and has limited the ability of physician's treatment. Staphylococcus aureus the most notorious pathogens causes morbidity and mortality especially in burn patients. However, Staphylococcus aureus rapidly acquired resistance to multiple antibiotics. Vancomycin, a glycopeptide antibiotic remains a drug of choice for treatment of severe Methicillin Resistance S. aureus infections. This study aimed to detect the emergence of beta-lactam and glycopeptide resistance genes. 50 clinical specimens of S. aureus collected from burn patients in burn and plastic surgery units in Sulaimani-Iraq city. All specimens were confirmed to be positive for S. aureus. All the isolates were assessed for their susceptibility to different antibiotics depending on NCCL standards, followed by Extended Spectrum Beta Lactamase detection by double disk diffusion synergy test. The production of ?- lactamases was evaluated in the isolated strains by several routine methods and polymerase chain reaction. Among the isolates 94% were Methicillin resistance and 34.28% were Extended Spectrum Beta Lactamase producer. PCR based molecular technique was done for the bla genes related to ?- lactamase enzymes by the specific primers, as well as genes which related to reduced sensitivity to Vancomycin were detected. The results indicated that all isolated showed the PBP1, PBP2, PBP3, PBP4, trfA and trfB, graSR, vraS except the vraR gene and the prolonged therapy of Methicillin resistance infection with teicoplanin have been associated with progress of resistance and the rise of tecoplanin resistance may be a prologue to evolving Vancomycin resistance. In conclusion, beta-lactam over taking can rise Vancomycin- Intermediate S. aureus strains leading to appearance of Vancomycin resistance although the treatment of Vancomycin resistant infections is challenging.

Cell division and cell wall synthesis in staphylococci need to be precisely coordinated and controlled to allow the cell to multiply while maintaining their nearly spherical shape. The mechanisms ensuring correct placement of the division plane and synthesis of new cell wall have been studied intensively, however, hitherto unknown factors and proteins are likely to play key roles in this complex interplay. We here identified and investigated a protein with major influence on cell morphology in Staphylococcus aureus. The protein, named SmdA (for staphylococcal morphology determinant A), is a membrane-protein with septum-enriched localization. By CRISPRi knockdown and overexpression combined with different microscopy techniques, we demonstrate that proper levels of SmdA is necessary for cell division, including septum formation and cell splitting. We also identified conserved residues in SmdA that are critical for its functionality. Pulldown- and bacterial two-hybrid interaction experiments showed that SmdA interacts with several known cell division- and cell wall synthesis proteins, including penicillin binding proteins (PBPs) and EzrA. Notably, SmdA also affects susceptibility to cell wall targeting antibiotics, particularly in methicillin-resistant S. aureus (MRSA). Together, our results show that S. aureus is dependent on balanced amounts of membrane-attached SmdA in order to carry out proper cell division.ImportanceStaphylococcus aureus is an important human and animal pathogen. Antibiotic resistance is a major problem in treatment of staphylococcal infections, and cell division and cell wall synthesis factors have previously been shown to modulate susceptibility to antibiotics in this species. In the current work we investigated the function of an essential protein named SmdA, which was identified based on its septal localization and knockdown phenotype resulting in defective cellular morphologies. We demonstrate that this protein is critical for normal cell division in S. aureus. Depletion of SmdA sensitize resistant staphylococci to β-lactam antibiotics. This work thus reveals a new staphylococcal cell division factor and a potential future target for narrow spectrum antimicrobials or compounds to resensitize antibiotic resistant staphylococcal strains.

Penicillin-binding proteins (PBPs) are a family of bacterial enzymes that are key components of cell-wall biosynthesis and the target of β-lactam antibiotics. Most microbial pathogens contain multiple structurally homologous PBP isoforms, making it difficult to target individual PBPs. To study the roles and regulation of specific PBP isoforms, a panel of bioorthogonal β-lactone probes was synthesized and compared. Fluorescent labeling confirmed selectivity, and PBPs were selectively enriched from Streptococcus pneumoniae lysates. Comparisons between fluorescent labeling of probes revealed that the accessibility of bioorthogonal reporter molecules to the bound probe in the native protein environment exerts a more significant effect on labeling intensity than the bioorthogonal reaction used, observations that are likely applicable beyond this class of probes or proteins. Selective, bioorthogonal activity-based probes for PBPs will facilitate the activity-based determination of the roles and regulation of specific PBP isoforms, a key gap in knowledge that has yet to be filled.

Bacterial cell division is mediated by a protein complex known as the divisome. Many protein–protein interactions in the divisome have been characterized. In this report, we analyse the role of the PASTA (Penicillin-binding protein And Serine Threonine kinase Associated) domains of Bacillus subtilis PBP2B. PBP2B itself is essential and cannot be deleted, but removing the PBP2B PASTA domains results in impaired cell division and a heat-sensitive phenotype. This resembles the deletion of divIB, a known interaction partner of PBP2B. Bacterial two-hybrid and co-immunoprecipitation analyses show that the interaction between PBP2B and DivIB is weakened when the PBP2B PASTA domains are removed. Combined, our results show that the PBP2B PASTA domains are required to strengthen the interaction between PBP2B and DivIB.

Invasive strains of Streptococcus pyogenes with significantly reduced susceptibility to β-lactam antibiotics have been recently described. These reports have caused considerable concern in the international infectious disease, medical microbiology, and public health communities because S. pyogenes has remained universally susceptible to β-lactam antibiotics for 70 years. Virtually all analyzed strains had single amino acid replacements in penicillin-binding protein 2X (PBP2X), a major target of β-lactam antibiotics in pathogenic bacteria. We used isogenic strains to test the hypothesis that a single amino acid replacement in PBP2X conferred a fitness advantage in a mouse model of necrotizing myositis. We determined that when mice were administered intermittent subtherapeutic dosing of benzylpenicillin, the strain with a Pro601Leu amino acid replacement in PBP2X that confers reduced β-lactam susceptibility in vitro was more fit, as assessed by the magnitude of colony-forming units recovered from disease tissue. These data provide important pathogenesis information that bears on this emerging global infectious disease problem.

The overuse of antibiotics has led to the emergence of multidrug-resistant pathogens. There is an urgent need to develop alternative therapeutic strategies to reduce mortality and morbidity related to drug-resistant bacterial infections. Self-synthesized tetrahedral framework nucleic acids (tFNAs) are used as the drug loading platform to deliver ampicillin to combat methicillin-resistant Staphylococcus aureus (MRSA) infection. The results of average dimension, zeta potential, transmission electron microscopy, and ultraviolet spectrophotometry showed that tFNAs-ampicillin combined with a sufficient encapsulation rate and good stability. tFNAs-ampicillin had a better affinity to MRSA than free ampicillin because it had a better uptake by MRSA cells. Additionally, tFNAs-ampicillin had a better antibacterial effect and lower levels of resistance development than free ampicillin. The downregulation of genes related to bacterial cell wall synthesis (murA and murZ) and upregulation of a gene related to antibiotic sensibility (PBP2) were responsible for the enhanced killing effect of tFNAs-ampicillin against MRSA.

The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs). ESC resistance is conferred by mosaic variants of penicillin-binding protein 2 (PBP2) that have diminished capacity to form acylated adducts with cephalosporins. To elucidate the molecular mechanisms of ESC resistance, we conducted a biochemical and high-resolution structural analysis of PBP2 variants derived from the decreased-susceptibility N. gonorrhoeae strain 35/02 and ESC-resistant strain H041. Our data reveal that mutations both lower affinity of PBP2 for ceftriaxone and restrict conformational changes that normally accompany acylation. Specifically, we observe that a G545S substitution hinders rotation of the β3 strand necessary to form the oxyanion hole for acylation and also traps ceftriaxone in a noncanonical configuration. In addition, F504L and N512Y substitutions appear to prevent bending of the β3–β4 loop that is required to contact the R1 group of ceftriaxone in the active site. Other mutations also appear to act by reducing flexibility in the protein. Overall, our findings reveal that restriction of protein dynamics in PBP2 underpins the ESC resistance of N. gonorrhoeae.

Machine learning techniques can be applied to MALDI-TOF mass spectral data of drug-treated cells to obtain classification models which assign the mechanism of action of drugs. Here, we present an example application of this concept to the screening of antibacterial drugs that act at the major bacterial target sites such as the ribosome, penicillin-binding proteins, and topoisomerases in a pharmacologically relevant phenotypic setting. We show that antibacterial effects can be identified and classified in a label-free, high-throughput manner using wild-type Escherichia coli and Staphylococcus aureus cells at variable levels of target engagement. This phenotypic approach, which combines mass spectrometry and machine learning, therefore denoted as PhenoMS-ML, may prove useful for the identification and development of novel antibacterial compounds and other pharmacological agents.

Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis inBacillus subtilis, we evaluated the PBP inhibition profiles of 21 β-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b or PBP4. Almost all penicillin scaffolds were co-selective for all or combinations of PBP2a, 2b and 4. Cephalosporins, on the other hand, possessed the lowest IC50values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin), 2b (cefotaxime) or 2a, 2b and 4 (cephalothin). Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid) and various co-selective inhibitors for other PBPs inB. subtiliswere discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for β-lactams and speculated to be involved in resistance inB. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.

Bacteria produce various D-amino acids, including non-canonical D-amino acids, to adapt to environmental changes and overcome a variety of threats. These D-amino acids are largely utilized as components of peptidoglycan, and they promote peptidoglycan remodeling and biofilm disassembly. The biosynthesis, maturation, and recycling of peptidoglycan are catalyzed by penicillin-binding proteins (PBPs). However, although non-canonical D-amino acids are known to be incorporated into peptidoglycan, the maturation and recycling of peptidoglycan containing such residues remain uncharacterized. Therefore, we investigated whether PBP4 and PBP5, low molecular mass (LMM) PBPs from Escherichia coli and Bacillus subtilis, are involved in these events of peptidoglycan metabolism. Enzyme assays using p-nitroaniline (pNA)-derivatized D-amino acids and peptidoglycan-mimicking peptides revealed that PBP4 and PBP5 from both species have peptidase activity toward substrates containing D-Asn, D-His, or D-Trp. These D-amino acids slowed the growth of dacA- or dacB-deficient E. coli (∆dacA or ∆dacB) relative to the wild-type strain. Additionally, these D-amino acids affected biofilm formation by the ∆dacB strain. Collectively, PBP4 and PBP5 are involved in the cleavage of peptidoglycan containing non-canonical D-amino acids, and these properties affect growth and biofilm formation.

Overuse and misuse of antibiotics leads to antibiotic resistance which has become a significant public health concern. Klebsiella pneumoniae is the most common pathogenic bacteria underlying nosocomial infections due to the expression of virulence factors and occurrence of antibiotic resistance. Evidence indicates that β-lactamase is involved in the antibiotic resistance of Klebsiella pneumoniae to β-lactam antibiotics. The aim of the present study was to investigate the association between the molecular biological mechanisms of antibiotic resistance of Klebsiella pneumoniae and extended-spectrum β-lactamase (ESBL). In order to assess temporal trends in prevalence and antimicrobial susceptibility, Klebsiella pneumoniae bacteria were isolated and the ESBL expression level was analyzed. Susceptibility tests were performed using automated systems. The β-lactam-resistance in Klebsiella pneumoniae was assessed by the β-lactam agar screen plate and respective MIC values were evaluated using E-test strips. The confirmatory disk diffusion methods were applied for phenotypic identification of the ESBL production of Klebsiella pneumoniae. The results showed that Klebsiella pneumoniae bacteria exhibited higher ESBL production after treatment with β-lactam compared to the control. The ESBL gene expression was upregulated in Klebsiella pneumoniae after treatment with β-lactam. Results identified that penicillin-binding proteins (PBPs) were associated with the growth and resistance to β-lactams. Zinc finger nuclease markedly inhibited the antibiotic resistance of Klebsiella pneumoniae to β-lactam. PBP knockdown abolished the inhibitory effects of zinc finger nuclease on the growth of Klebsiella pneumonia e induced by β-lactam antibiotic treatment. In conclusion, these results suggest that the resistance of Klebsiella pneumoniae bacteria to antimicrobial drugs is through the ESBL signaling pathway, which indicates that ESBL may be a potential target for abolishing resistance to β-lactam.

Molecular probes typically require structural modifications to allow for the immobilisation or bioconjugation with a desired substrate but the effects of these changes are often not evaluated. Here, we set out to determine the effects of attaching functional handles to a first-generation cephalosporin. A series of cephalexin derivatives was prepared, equipped with chemical tethers suitable for the site-selective conjugation of antibiotics to functionalised surfaces. The tethers were positioned remotely from the β-lactam ring to ensure minimal effect to the antibiotic's pharmacophore. Herein, the activity of the modified antibiotics was evaluated for binding to the therapeutic target, the penicillin binding proteins, and shown to maintain binding interactions. In addition, the deactivation of the modified drugs by four β-lactamases (TEM-1, CTX-M-15, AmpC, NDM-1) was investigated and the effect of the tethers on the catalytic efficiencies determined. CTX-M-15 was found to favour hydrolysis of the parent antibiotic without a tether, whereas AmpC and NDM-1 were found to favour the modified analogues. Furthermore, the antimicrobial activity of the derivatives was evaluated to investigate the effect of the structural modifications on the antimicrobial activity of the parent drug, cephalexin.