Staphylococcus succinus Infective Endocarditis, France.

Infective endocarditis is a rare condition in humans and is associated with high illness and death rates. We describe a case of infective endocarditis caused by Staphylococcus succinus bacteria in France. We used several techniques for susceptibility testing for this case to determine the oxacillin profile.

Sensitizing methicillin-resistant Staphylococcus aureus (MRSA) to cefuroxime: the synergic effect of bicarbonate and the wall teichoic acid inhibitor ticlopidine.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a major challenge for clinicians due, in part, to their resistance to most ß-lactams, the first-line treatment for methicillin-susceptible S. aureus. A phenotype termed "NaHCO3-responsiveness" has been identified, wherein many clinical MRSA isolates are rendered susceptible to standard-of-care ß-lactams in the presence of physiologically relevant concentrations of NaHCO3, in vitro and ex vivo; moreover, such "NaHCO3-responsive" isolates can be effectively cleared by ß-lactams from target tissues in experimental infective endocarditis (IE). One mechanistic impact of NaHCO3 exposure on NaHCO3-responsive MRSA is to repress WTA synthesis. This NaHCO3 effect mimics the phenotype of tarO-deficient MRSA, including sensitization to the PBP2-targeting ß-lactam, cefuroxime (CFX). Herein, we further investigated the impacts of NaHCO3 exposure on CFX susceptibility in the presence and absence of a WTA synthesis inhibitor, ticlopidine (TCP), in a collection of clinical MRSA isolates from skin and soft tissue infections (SSTI) and bloodstream infections (BSI). NaHCO3 and/or TCP enhanced susceptibility to CFX in vitro, by both minimum inhibitor concentration (MIC) and time-kill assays, as well as in an ex vivo simulated endocarditis vegetations (SEV) model, in NaHCO3-responsive MRSA. Furthermore, in experimental IE (presumably in the presence of endogenous NaHCO3), pre-exposure to TCP prior to infection sensitized the NaHCO3-responsive MRSA strain (but not the non-responsive strain) to enhanced clearances by CFX in target tissues. These data support the notion that NaHCO3 is acting similarly to WTA synthesis inhibitors, and that such inhibitors have potential translational applications in the treatment of certain MRSA strains in conjunction with specific ß-lactam agents.

Penicillin-binding protein redundancy in Bacillus subtilis enables growth during alkaline shock.

Penicillin-binding proteins (PBPs) play critical roles in cell wall construction, cell shape maintenance, and bacterial replication. Bacteria maintain a diversity of PBPs, indicating that despite their apparent functional redundancy, there is differentiation across the PBP family. Apparently-redundant proteins can be important for enabling an organism to cope with environmental stressors. In this study, we evaluated the consequence of environmental pH on PBP enzymatic activity in Bacillus subtilis. Our data show that a subset of PBPs in B. subtilis change activity levels during alkaline shock and that one PBP isoform is rapidly modified to generate a smaller protein (i.e., PBP1a to PBP1b). Our results indicate that a subset of the PBPs are favored for growth under alkaline conditions, while others are readily dispensable. Indeed, we found that this phenomenon could also be observed in Streptococcus pneumoniae, implying that it may be generalizable across additional bacterial species and further emphasizing the evolutionary benefit of maintaining many, seemingly-redundant periplasmic enzymes.IMPORTANCEMicrobes adapt to ever-changing environments and thrive over a vast range of conditions. While bacterial genomes are relatively small, significant portions encode for "redundant" functions. Apparent redundancy is especially pervasive in bacterial proteins that reside outside of the inner membrane. While conditions within the cytoplasm are carefully controlled, those of the periplasmic space are largely determined by the cell's exterior environment. As a result, proteins within this environmentally exposed region must be capable of functioning under a vast array of conditions, and/or there must be several similar proteins that have evolved to function under a variety of conditions. This study examines the activity of a class of enzymes that is essential in cell wall construction to determine if individual proteins might be adapted for activity under particular growth conditions. Our results indicate that a subset of these proteins are preferred for growth under alkaline conditions, while others are readily dispensable.

Boronic acid inhibitors of penicillin-binding protein 1b: serine and lysine labelling agents.

Penicillin-binding proteins (PBPs) contribute to bacterial cell wall biosynthesis and are targets of antibacterial agents. Here, we investigated PBP1b inhibition by boronic acid derivatives. Chemical starting points were identified by structure-based virtual screening and aliphatic boronic acids were selected for further investigations. Structure-activity relationship studies focusing on the branching of the boron-connecting carbon and quantum mechanical/molecular mechanical simulations showed that reaction barrier free energies are compatible with fast reversible covalent binding and small or missing reaction free energies limit the inhibitory activity of the investigated boronic acid derivatives. Therefore, covalent labelling of the lysine residue of the catalytic dyad was also investigated. Compounds with a carbonyl warhead and an appropriately positioned boronic acid moiety were shown to inhibit and covalently label PBP1b. Reversible covalent labelling of the catalytic lysine by imine formation and the stabilisation of the imine by dative N-B bond is a new strategy for PBP1b inhibition.

Penicillin-Binding Protein 5/6 Acting as a Decoy Target in Pseudomonas aeruginosa Identified by Whole-Cell Receptor Binding and Quantitative Systems Pharmacology.

The ß-lactam antibiotics have been successfully used for decades to combat susceptible Pseudomonas aeruginosa, which has a notoriously difficult to penetrate outer membrane (OM). However, there is a dearth of data on target site penetration and covalent binding of penicillin-binding proteins (PBP) for ß-lactams and ß-lactamase inhibitors in intact bacteria. We aimed to determine the time course of PBP binding in intact and lysed cells and estimate the target site penetration and PBP access for 15 compounds in P. aeruginosa PAO1. All ß-lactams (at 2 × MIC) considerably bound PBPs 1 to 4 in lysed bacteria. However, PBP binding in intact bacteria was substantially attenuated for slow but not for rapid penetrating ß-lactams. Imipenem yielded 1.5 ± 0.11 log10 killing at 1h compared to <0.5 log10 killing for all other drugs. Relative to imipenem, the rate of net influx and PBP access was ~ 2-fold slower for doripenem and meropenem, 7.6-fold for avibactam, 14-fold for ceftazidime, 45-fold for cefepime, 50-fold for sulbactam, 72-fold for ertapenem, ~ 249-fold for piperacillin and aztreonam, 358-fold for tazobactam, ~547-fold for carbenicillin and ticarcillin, and 1,019-fold for cefoxitin. At 2 × MIC, the extent of PBP5/6 binding was highly correlated (r2 = 0.96) with the rate of net influx and PBP access, suggesting that PBP5/6 acted as a decoy target that should be avoided by slowly penetrating, future ß-lactams. This first comprehensive assessment of the time course of PBP binding in intact and lysed P. aeruginosa explained why only imipenem killed rapidly. The developed novel covalent binding assay in intact bacteria accounts for all expressed resistance mechanisms.

PBP2b Mutations Improve the Growth of Phage-Resistant Lactococcus cremoris Lacking Polysaccharide Pellicle.

Lactococcus lactis and Lactococcus cremoris are Gram-positive lactic acid bacteria widely used as starter in milk fermentations. Lactococcal cells are covered with a polysaccharide pellicle (PSP) that was previously shown to act as the receptor for numerous bacteriophages of the Caudoviricetes class. Thus, mutant strains lacking PSP are phage resistant. However, because PSP is a key cell wall component, PSP-negative mutants exhibit dramatic alterations of cell shape and severe growth defects, which limit their technological value. In the present study, we isolated spontaneous mutants with improved growth, from L. cremoris PSP-negative mutants. These mutants grow at rates similar to the wild-type strain, and based on transmission electron microscopy analysis, they exhibit improved cell morphology compared to their parental PSP-negative mutants. In addition, the selected mutants maintain their phage resistance. Whole-genome sequencing of several such mutants showed that they carried a mutation in pbp2b, a gene encoding a penicillin-binding protein involved in peptidoglycan biosynthesis. Our results indicate that lowering or turning off PBP2b activity suppresses the requirement for PSP and ameliorates substantially bacterial fitness and morphology. IMPORTANCE Lactococcus lactis and Lactococcus cremoris are widely used in the dairy industry as a starter culture. As such, they are consistently challenged by bacteriophage infections which may result in reduced or failed milk acidification with associated economic losses. Bacteriophage infection starts with the recognition of a receptor at the cell surface, which was shown to be a cell wall polysaccharide (the polysaccharide pellicle [PSP]) for the majority of lactococcal phages. Lactococcal mutants devoid of PSP exhibit phage resistance but also reduced fitness, since their morphology and division are severely impaired. Here, we isolated spontaneous, food-grade non-PSP-producing L. cremoris mutants resistant to bacteriophage infection with a restored fitness. This study provides an approach to isolate non-GMO phage-resistant L. cremoris and L. lactis strains, which can be applied to strains with technological functionalities. Also, our results highlight for the first time the link between peptidoglycan and cell wall polysaccharide biosynthesis.

Antibacterial activity and synergistic antibiotic mechanism of trialdehyde phloroglucinol against methicillin-resistant Staphylococcus aureus.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has become a critical global concern. Identifying new anti-S. aureus agents or therapeutic strategies are urgently needed to treat S. aureus infection. The present study investigated the antibacterial activity of 16 phenolic compounds against MRSA, four of which exhibited antibacterial activity. Their antibacterial activities increased in a dose-dependent manner but showed different responses with the extension of treatment time. Trialdehyde phloroglucinol (TPG) and 2-nitrophloroglucinol (NPG) maintained stable antibacterial activity; however, that of dichlorophenol and myricetin decreased rapidly over 24 hr of treatment. Checkerboard and time-kill assays indicated that TPG and NPG exhibited strong synergistic antibacterial activities with penicillin or bacitracin. Microscopic observation and membrane integrity analysis showed that the combination of TPG and penicillin destroyed the MRSA cell membrane, resulting in the leakage of intracellular biomacromolecules, marked changes in surface zeta potential, and the collapse of membrane potential. Moreover, the combination significantly decreased penicillinase activity and penicillin-binding protein 2a mRNA expression, inhibiting MRSA growth. Taken together, these results demonstrated that the combination of the phloroglucinol derivative TPG and penicillin has significant synergistic anti-MRSA activity and can serve as a potential therapeutic strategy to treat MRSA infections.

Deletion of pbpC Enhances Bacterial Pathogenicity on Tomato by Affecting Biofilm Formation, Exopolysaccharides Production, and Exoenzyme Activities in Clavibacter michiganensis.

Penicillin-binding proteins (PBPs) are considered essential for bacterial peptidoglycan biosynthesis and cell wall assembly. Clavibacter michiganensis is a representative Gram-positive bacterial species that causes bacterial canker in tomato. pbpC plays a significant role in maintaining cell morphological characteristics and stress responses in C. michiganensis. The current study demonstrated that the deletion of pbpC commonly enhances bacterial pathogenicity in C. michiganensis and revealed the mechanisms through which this occurs. The expression of interrelated virulence genes, including celA, xysA, xysB, and pelA, were significantly upregulated in △pbpC mutants. Compared with those in wild-type strains, exoenzyme activities, the formation of biofilm, and the production of exopolysaccharides (EPS) were significantly increased in △pbpC mutants. It is noteworthy that EPS were responsible for the enhancement in bacterial pathogenicity, with the degree of necrotic tomato stem cankers intensifying with the injection of a gradient of EPS from C. michiganensis. These findings highlight new insights into the role of pbpC affecting bacterial pathogenicity, with an emphasis on EPS, advancing the current understanding of phytopathogenic infection strategies for Gram-positive bacteria.

Organization of peptidoglycan synthesis in nodes and separate rings at different stages of cell division of Streptococcus pneumoniae.

Bacterial peptidoglycan (PG) synthesis requires strict spatiotemporal organization to reproduce specific cell shapes. In ovoid-shaped Streptococcus pneumoniae (Spn), septal and peripheral (elongation) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D-SIM (structured-illumination microscopy). Labeling with fluorescent D-amino acids (FDAA) showed that areas of new transpeptidase (TP) activity catalyzed by penicillin-binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading-edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA-marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Pulses of FDAA labeling revealed an arrangement of separate regularly spaced "nodes" of TP activity around the division site of predivisional cells. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division.

Effects of Inactivation of d,d-Transpeptidases of Acinetobacter baumannii on Bacterial Growth and Susceptibility to ß-Lactam Antibiotics.

Resistance to ß-lactams, the most used antibiotics worldwide, constitutes the major problem for the treatment of bacterial infections. In the nosocomial pathogen Acinetobacter baumannii, ß-lactamase-mediated resistance to the carbapenem family of ß-lactam antibiotics has resulted in the selection and dissemination of multidrug-resistant isolates, which often cause infections characterized by high mortality rates. There is thus an urgent demand for new ß-lactamase-resistant antibiotics that also inhibit their targets, penicillin-binding proteins (PBPs). As some PBPs are indispensable for the biosynthesis of the bacterial cell wall and survival, we evaluated their importance for the growth of A. baumannii by performing gene inactivation studies of d,d-transpeptidase domains of high-molecular-mass (HMM) PBPs individually and in combination with one another. We show that PBP3 is essential for A. baumannii survival, as deletion mutants of this d,d-transpeptidase were not viable. The inactivation of PBP1a resulted in partial cell lysis and retardation of bacterial growth, and these effects were further enhanced by the additional inactivation of PBP2 but not PBP1b. Susceptibility to ß-lactam antibiotics increased 4- to 8-fold for the A. baumannii PBP1a/PBP1b/PBP2 triple mutant and 2- to 4-fold for all remaining mutants. Analysis of the peptidoglycan structure revealed a significant change in the muropeptide composition of the triple mutant and demonstrated that the lack of d,d-transpeptidase activity of PBP1a, PBP1b, and PBP2 is compensated for by an increase in the l,d-transpeptidase-mediated cross-linking activity of LdtJ. Overall, our data showed that in addition to essential PBP3, the simultaneous inhibition of PBP1a and PBP2 or PBPs in combination with LdtJ could represent potential strategies for the design of novel drugs against A. baumannii.

Expanded profiling of ß-lactam selectivity for penicillin-binding proteins in Streptococcus pneumoniae D39.

Penicillin-binding proteins (PBPs) are integral to bacterial cell division as they mediate the final steps of cell wall maturation. Selective fluorescent probes are useful for understanding the role of individual PBPs, including their localization and activity during growth and division of bacteria. For the development of new selective probes for PBP imaging, several ß-lactam antibiotics were screened, as they are known to covalently bind PBP in vivo. The PBP inhibition profiles of 16 commercially available ß-lactam antibiotics were evaluated in an unencapsulated derivative of the D39 strain of Streptococcus pneumoniae, IU1945. These ß-lactams have not previously been characterized for their PBP inhibition profiles in S. pneumoniae and these data augment those obtained from a library of 20 compounds that we previously reported. We investigated seven penicillins, three carbapenems, and six cephalosporins. Most of these ß-lactams were found to be co-selective for PBP2x and PBP3, as was noted in our previous studies. Six out of 16 antibiotics were selective for PBP3 and one molecule was co-selective for PBP1a and PBP3. Overall, this work expands the chemical space available for development of future ß-lactam-based probes for specific pneumococcal PBP labeling and these methods can be used for the development of probes for PBP labelling in other bacterial species.

Identification and molecular characterization of penicillin-nonsusceptible Streptococcus pneumoniae isolates recovered from invasive infections in a pre-pneumococcal vaccine era.

BACKGROUND: Given the significant role of penicillin-nonsusceptible Streptococcus pneumoniae in inducing severe infectious diseases, identifying serotypes and genotypes that can mediate antimicrobial resistance has become a pillar of treatment strategies. This study aims to determine the correlation between the minimum inhibitory concentration of antimicrobial agents and amino acid mutations in penicillin-binding proteins. Moreover, molecular serotyping and multiple-locus variable number tandem repeat analysis typing were first-ever performed to characterize the invasive penicillin-nonsusceptible S. pneumoniae isolates in Iran. METHODS: Of 149 isolates, antimicrobial susceptibility tests were performed against penicillin, ceftriaxone, and cefotaxime by the MIC Test Strip, and sequence analysis of the pbp genes was performed through PCR-sequencing method. All penicillin-nonsusceptible S. pneumoniae isolates were serotyped and genotyped by sequential multiplex PCR and multiple-locus variable-number tandem repeat analysis, respectively. RESULTS: Among pneumococcal isolates, 53 isolates were classified as penicillin-nonsusceptible S. pneumoniae, of which 38 (71.7%) and 15 (28.3%) were resistant and intermediate to penicillin, respectively. Furthermore, ceftriaxone- and cefotaxime-nonsusceptible pneumococci constituted 33 (62.2%) and 29 cases (54.7%), respectively. Of note, there were 8 and 41 different serotypes and multiple-locus variable-number tandem repeat analysis types, respectively. CONCLUSIONS: Due to the increasing resistance to antimicrobial agents, the most efficient approach to preventing pneumococcal infection mortality as vaccine-preventable diseases is focusing on wide-spectrum vaccination. Based on our findings, the 13-valent pneumococcal conjugate vaccine could considerably reduce the incidence of invasive pneumococcal diseases due to the high rate of serotype coverage.

Penicillin-Binding Proteins, ß-Lactamases, and ß-Lactamase Inhibitors in ß-Lactam-Producing Actinobacteria: Self-Resistance Mechanisms.

The human society faces a serious problem due to the widespread resistance to antibiotics in clinical practice. Most antibiotic biosynthesis gene clusters in actinobacteria contain genes for intrinsic self-resistance to the produced antibiotics, and it has been proposed that the antibiotic resistance genes in pathogenic bacteria originated in antibiotic-producing microorganisms. The model actinobacteria Streptomyces clavuligerus produces the ß-lactam antibiotic cephamycin C, a class A ß-lactamase, and the ß lactamases inhibitor clavulanic acid, all of which are encoded in a gene supercluster; in addition, it synthesizes the ß-lactamase inhibitory protein BLIP. The secreted clavulanic acid has a synergistic effect with the cephamycin produced by the same strain in the fight against competing microorganisms in its natural habitat. High levels of resistance to cephamycin/cephalosporin in actinobacteria are due to the presence (in their ß-lactam clusters) of genes encoding PBPs which bind penicillins but not cephalosporins. We have revised the previously reported cephamycin C and clavulanic acid gene clusters and, in addition, we have searched for novel ß-lactam gene clusters in protein databases. Notably, in S. clavuligerus and Nocardia lactamdurans, the ß-lactamases are retained in the cell wall and do not affect the intracellular formation of isopenicillin N/penicillin N. The activity of the ß-lactamase in S. clavuligerus may be modulated by the ß-lactamase inhibitory protein BLIP at the cell-wall level. Analysis of the ß-lactam cluster in actinobacteria suggests that these clusters have been moved by horizontal gene transfer between different actinobacteria and have culminated in S. clavuligerus with the organization of an elaborated set of genes designed for fine tuning of antibiotic resistance and cell wall remodeling for the survival of this Streptomyces species. This article is focused specifically on the enigmatic connection between ß-lactam biosynthesis and ß-lactam resistance mechanisms in the producer actinobacteria.

Beta-lactam antibiotics and viridans group streptococci.

The aim of this review is to present an update on the susceptibility of viridans group streptococci (VGS) to ß-lactam antimicrobials, with emphasis on the Argentinean scenario. VGS are a heterogeneous group including five groups of species, each one exhibiting peculiar susceptibility patterns to penicillin (PEN). Species of the Streptococcus mitis group are frequently nonsusceptible to PEN. PEN resistance is associated with changes in PEN-binding proteins. In Argentina, one to two thirds of VGS are nonsusceptible to PEN. Third generation cephalosporins and carbapenems are currently more effective in vitro than PEN against VGS. Mortality was associated to nonsusceptibility to PEN in at least two studies involving patients with bacteremia caused by VGS. Treatment of endocarditis due to VGS should be adjusted/to the PEN susceptibility of the isolates. Vancomycin may be an alternative choice for treating endocarditis caused by PEN-resistant isolates (MIC≥4µg/ml).

Functional Insights into the High-Molecular-Mass Penicillin-Binding Proteins of Streptococcus agalactiae Revealed by Gene Deletion and Transposon Mutagenesis Analysis.

High-molecular-mass penicillin-binding proteins (PBPs) are enzymes that catalyze the biosynthesis of bacterial cell wall peptidoglycan. The Gram-positive bacterial pathogen Streptococcus agalactiae (group B streptococcus [GBS]) produces five high-molecular-mass PBPs, namely, PBP1A, PBP1B, PBP2A, PBP2B, and PBP2X. Among these, only PBP2X is essential for cell viability, whereas the other four PBPs are individually dispensable. The biological function of the four nonessential PBPs is poorly characterized in GBS. We deleted the pbp1a, pbp1b, pbp2a, and pbp2b genes individually from a genetically well-characterized serotype V GBS strain and studied the phenotypes of the four isogenic mutant strains. Compared to the wild-type parental strain, (i) none of the pbp isogenic mutant strains had a significant growth defect in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY) rich medium, (ii) isogenic mutant Δpbp1a and Δpbp1b strains had significantly increased susceptibility to penicillin and ampicillin, and (iii) isogenic mutant Δpbp1a and Δpbp2b strains had significantly longer chain lengths. Using saturated transposon mutagenesis and transposon insertion site sequencing, we determined the genes essential for the viability of the wild-type GBS strain and each of the four isogenic pbp deletion mutant strains in THY rich medium. The pbp1a gene is essential for cell viability in the pbp2b deletion background. Reciprocally, pbp2b is essential in the pbp1a deletion background. Moreover, the gene encoding RodA, a peptidoglycan polymerase that works in conjunction with PBP2B, is also essential in the pbp1a deletion background. Together, our results suggest functional overlap between PBP1A and the PBP2B-RodA complex in GBS cell wall peptidoglycan biosynthesis. IMPORTANCE High-molecular-mass penicillin-binding proteins (HMM PBPs) are enzymes required for bacterial cell wall biosynthesis. Bacterial pathogen group B streptococcus (GBS) produces five distinct HMM PBPs. The biological functions of these proteins are not well characterized in GBS. In this study, we performed a comprehensive deletion analysis of genes encoding HMM PBPs in GBS. We found that deleting certain PBP-encoding genes altered bacterial susceptibility to beta-lactam antibiotics, cell morphology, and the essentiality of other enzymes involved in cell wall peptidoglycan synthesis. The results of our study shed new light on the biological functions of PBPs in GBS.

Impact of FtsZ Inhibition on the Localization of the Penicillin Binding Proteins in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen of acute clinical importance. Combination treatment with an FtsZ inhibitor potentiates the activity of penicillin binding protein (PBP)-targeting ß-lactam antibiotics against MRSA. To explore the mechanism underlying this synergistic behavior, we examined the impact of treatment with the FtsZ inhibitor TXA707 on the spatial localization of the five PBP proteins expressed in MRSA. In the absence of drug treatment, PBP1, PBP2, PBP3, and PBP4 colocalize with FtsZ at the septum, contributing to new cell wall formation. In contrast, PBP2a localizes to distinct foci along the cell periphery. Upon treatment with TXA707, septum formation becomes disrupted, and FtsZ relocalizes away from midcell. PBP1 and PBP3 remain significantly colocalized with FtsZ, while PBP2, PBP4, and PBP2a localize away from FtsZ to specific sites along the periphery of the enlarged cells. We also examined the impact on PBP2a and PBP2 localization of treatment with ß-lactam antibiotic oxacillin alone and in synergistic combination with TXA707. Significantly, PBP2a localizes to the septum in approximately 15% of the oxacillin-treated cells, a behavior that likely contributes to the ß-lactam resistance of MRSA. Combination treatment with TXA707 causes both PBP2a and PBP2 to localize in malformed septum-like structures. Our collective results suggest that PBP2, PBP4, and PBP2a may function collaboratively in peripheral cell wall repair and maintenance in response to FtsZ inhibition by TXA707. Cotreatment with oxacillin appears to reduce the availability of PBP2a to assist in this repair, thereby rendering the MRSA cells more susceptible to the ß-lactam. IMPORTANCE MRSA is a multidrug-resistant bacterial pathogen of acute clinical importance, infecting many thousands of individuals globally each year. The essential cell division protein FtsZ has been identified as an appealing target for the development of new drugs to combat MRSA infections. Through synergistic actions, FtsZ-targeting agents can sensitize MRSA to antibiotics like the ß-lactams that would otherwise be ineffective. This study provides key insights into the mechanism underlying this synergistic behavior as well as MRSA resistance to ß-lactam drugs. The results of this work will help guide the identification and optimization of combination drug regimens that can effectively treat MRSA infections and reduce the potential for future resistance.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation.

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

Comparison of Bioorthogonal ß-Lactone Activity-Based Probes for Selective Labeling of Penicillin-Binding Proteins.

Penicillin-binding proteins (PBPs) are a family of bacterial enzymes that are key components of cell-wall biosynthesis and the target of ß-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 ß-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.

Comparative genomics of invasive Streptococcus pneumoniae CC320/271 serotype 19F/19A before the introduction of pneumococcal vaccine in India.

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.

Molecular Basis of AmpC ß-Lactamase Induction by Avibactam in Pseudomonas aeruginosa: PBP Occupancy, Live Cell Binding Dynamics and Impact on Resistant Clinical Isolates Harboring PDC-X Variants.

Avibactam belongs to the new class of diazabicyclooctane ß-lactamase inhibitors. Its inhibitory spectrum includes class A, C and D enzymes, including P. aeruginosa AmpC. Nonetheless, recent reports have revealed strain-dependent avibactam AmpC induction. In the present work, we wanted to assess the mechanistic basis underlying AmpC induction and determine if derepressed PDC-X mutated enzymes from ceftazidime/avibactam-resistant clinical isolates were further inducible. We determined avibactam concentrations that half-maximally inhibited (IC50) bocillin FL binding. Inducer ß-lactams were also studied as comparators. Live cells' time-course penicillin-binding proteins (PBPs) occupancy of avibactam was studied. To assess the ampC induction capacity of avibactam and comparators, qRT-PCR was performed in wild-type PAO1, PBP4, triple PBP4, 5/6 and 7 knockout derivatives and two ceftazidime/avibactam-susceptible/resistant XDR clinical isolates belonging to the epidemic high-risk clone ST175. PBP4 inhibition was observed for avibactam and ß-lactam comparators. Induction capacity was consistently correlated with PBP4 binding affinity. Outer membrane permeability-limited PBP4 binding was observed in the live cells' assay. As expected, imipenem and cefoxitin showed strong induction in PAO1, especially for carbapenem; avibactam induction was conversely weaker. Overall, the inducer effect was less remarkable in ampC-derepressed mutants and nonetheless absent upon avibactam exposure in the clinical isolates harboring mutated AmpC variants and their parental strains.