Evolving standards of care and new challenges in the management of HER2-positive breast cancer.

The management of human epidermal growth factor receptor (HER2)-positive breast cancer (BC) has rapidly evolved over the last 20 years. Major advances have led to US Food and Drug Administration approval of 7 HER2-targeted therapies for the treatment of early-stage and/or advanced-stage disease. Although oncologic outcomes continue to improve, most patients with advanced HER2-positive BC ultimately die of their disease because of primary or acquired resistance to therapy, and patients with HER2-positive early BC who have residual invasive disease after preoperative systemic therapy are at a higher risk of distant recurrence and death. The concept of treatment de-escalation and escalation is increasingly important to optimally tailor therapy for patients with HER2-positive BC and is a major focus of the current review. Research efforts in this regard are discussed as well as updates regarding the evolving standard of care in the (neo)adjuvant and metastatic settings, including the use of novel combination therapies. The authors also briefly discuss ongoing challenges in the management of HER2-positive BC (eg, intrinsic vs acquired drug resistance, the identification of predictive biomarkers, the integration of imaging techniques to guide clinical practice), and the treatment of HER2-positive brain metastases. Research aimed at superseding these challenges will be imperative to ensure continued progress in the management of HER2-positive BC going forward.

Antibiotic Resistances of Clostridioides difficile.

The rapid evolution of antibiotic resistance in Clostridioides difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are a matter of concern for public health. Antibiotic resistance plays an important role in driving C. difficile epidemiology. Emergence of new types is often associated with the emergence of new resistances, and most of the epidemic C. difficile clinical isolates is currently resistant to multiple antibiotics. In particular, it is to worth to note the recent identification of strains with reduced susceptibility to the first-line antibiotics for CDI treatment and/or for relapsing infections. Antibiotic resistance in C. difficile has a multifactorial nature. Acquisition of genetic elements and alterations of the antibiotic target sites, as well as other factors, such as variations in the metabolic pathways or biofilm production, contribute to the survival of this pathogen in the presence of antibiotics. Different transfer mechanisms facilitate the spread of mobile elements among C. difficile strains and between C. difficile and other species. Furthermore, data indicate that both genetic elements and alterations in the antibiotic targets can be maintained in C. difficile regardless of the burden imposed on fitness, and therefore resistances may persist in C. difficile population in absence of antibiotic selective pressure.

Understanding the Evolution and Transmission Dynamics of Antibiotic Resistance Genes: A Comprehensive Review.

Antibiotic resistance poses a formidable challenge to global public health, necessitating comprehensive understanding and strategic interventions. This review explores the evolution and transmission dynamics of antibiotic resistance genes, with a focus on Bangladesh. The indiscriminate use of antibiotics, compounded by substandard formulations and clinical misdiagnosis, fuels the emergence and spread of resistance in the country. Studies reveal high resistance rates among common pathogens, emphasizing the urgent need for targeted interventions and rational antibiotic use. Molecular assessments uncover a diverse array of antibiotic resistance genes in environmental reservoirs, highlighting the complex interplay between human activities and resistance dissemination. Horizontal gene transfer mechanisms, particularly plasmid-mediated conjugation, facilitate the exchange of resistance determinants among bacterial populations, driving the evolution of multidrug-resistant strains. The review discusses clinical implications, emphasizing the interconnectedness of environmental and clinical settings in resistance dynamics. Furthermore, bioinformatic and experimental evidence elucidates novel mechanisms of resistance gene transfer, underscoring the dynamic nature of resistance evolution. In conclusion, combating antibiotic resistance requires a multifaceted approach, integrating surveillance, stewardship, and innovative research to preserve the efficacy of antimicrobial agents and safeguard public health.

Repeated Emergence of Variant TetR Family Regulator, FarR, and Increased Resistance to Antimicrobial Unsaturated Fatty Acid among Clonal Complex 5 Methicillin-Resistant Staphylococcus aureus.

Resistance-nodulation-division (RND) superfamily efflux pumps promote antibiotic resistance in Gram-negative pathogens, but their role in Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) is undocumented. However, recent in vitro selections for resistance of S. aureus to an antimicrobial fatty acid, linoleic acid, and an antibiotic, rhodomyrtone, identified H121Y and C116R substitution variants, respectively, in a TetR family regulator, FarR, promoting increased expression of the RND pump FarE. Hypothesizing that in vivo selection pressures have also promoted the emergence of FarR variants, we searched available genome data and found that strains with FarRH121Y from human and bovine hosts have emerged sporadically in clonal complexes (CCs) CC1, CC30, CC8, CC22, and CC97, whereas multiple FarR variants have occurred within CC5 hospital-associated (HA)-MRSA. Of these, FarRE160G and FarRE93EE were exclusive to CC5, while FarRC116Y, FarRP165L, and FarRG166D also occurred in nonrelated CCs, primarily from bovine hosts. Within CC5, FarRC116Y and FarRG166D strains were polyphyletic, each exhibiting two emergence events. FarRC116Y and FarRE160G were individually sufficient to confer increased expression of FarE and enhanced resistance to linoleic acid (LA). Isolates with FarRE93EE were most closely related to S. aureus N315 MRSA and exhibited increased resistance independently of FarRE93EE. Accumulation of pseudogenes and additional polymorphisms in FarRE93EE strains contributed to a multiresistance phenotype which included fosfomycin and fusidic acid resistance in addition to increased linoleic acid resistance. These findings underscore the remarkable adaptive capacity of CC5 MRSA, which includes the polyphyletic USA100 lineage of HA-MRSA that is endemic in the Western hemisphere and known for the acquisition of multiple resistance phenotypes.

Resistance to prostate cancer treatments.

A review of the current treatment options for prostate cancer and the formation of resistance to these regimens has been compiled including primary, acquired, and cross-resistance. The diversification of the pathways involved and the escape routes the tumor is utilizing have been addressed. Whereas early stages of tumor can be cured, there is no treatment available after a point of no return has been reached, leaving palliative treatment as the only option. The major reasons for this outcome are the heterogeneity of tumors, both inter- and intra-individually and the nearly endless number of escape routes, which the tumor can select to overcome the effects of treatment. This means that more focus should be applied to the individualization of both diagnosis and therapy of prostate cancer. In addition to current treatment options, novel drugs and ongoing clinical trials have been addressed in this review.

Antibiotic Resistance in Dermatology Part 1: Mechanisms of Resistance.

Virtually any antibiotic can be used in dermatology given the broad range of conditions treated. With the widespread use of antibiotics and the rapid emergence of resistant organisms, it is important to understand the mechanisms at play that contribute to resistance.

Selection of AmpC ß-Lactamase Variants and Metallo-ß-Lactamases Leading to Ceftolozane/Tazobactam and Ceftazidime/Avibactam Resistance during Treatment of MDR/XDR Pseudomonas aeruginosa Infections.

Infections caused by ceftolozane-tazobactam and ceftazidime-avibactam-resistant P. aeruginosa infections are an emerging concern. We aimed to analyze the underlying ceftolozane-tazobactam and ceftazidime-avibactam resistance mechanisms in all multidrug-resistant or extensively drug-resistant (MDR/XDR) P. aeruginosa isolates recovered during 1 year (2020) from patients with a documented P. aeruginosa infection. Fifteen isolates showing ceftolozane-tazobactam and ceftazidime-avibactam resistance were evaluated. Clinical conditions, previous positive cultures, and ß-lactams received in the previous month were reviewed for each patient. MICs were determined by broth microdilution. Multilocus sequence types (MLSTs) and resistance mechanisms were determined using short- and long-read whole-genome sequencing (WGS). The impact of Pseudomonas-derived cephalosporinases (PDCs) on ß-lactam resistance was demonstrated by cloning into an ampC-deficient PAO1 derivative (PAOΔC) and construction of 3D models. Genetic support of acquired ß-lactamases was determined in silico from high-quality hybrid assemblies. In most cases, the isolates were recovered after treatment with ceftolozane-tazobactam or ceftazidime-avibactam. Seven isolates from different sequence types (STs) owed their ß-lactam resistance to chromosomal mutations and all displayed specific substitutions in PDC: Phe121Leu and Gly222Ser, Pro154Leu, Ala201Thr, Gly214Arg, ΔGly203-Glu219, and Glu219Lys. In the other eight isolates, the ST175 clone was overrepresented (6 isolates) and associated with IMP-28 and IMP-13, whereas two ST1284 isolates produced VIM-2. The cloned PDCs conferred enhanced cephalosporin resistance. The 3D PDC models revealed rearrangements affecting residues involved in cephalosporin hydrolysis. Carbapenemases were chromosomal (VIM-2) or plasmid-borne (IMP-28, IMP-13) and associated with class-1 integrons located in Tn402-like transposition modules. Our findings highlighted that cephalosporin/ß-lactamase inhibitors are potential selectors of MDR/XDR P. aeruginosa strains producing PDC variants or metallo-ß-lactamases. Judicious use of these agents is encouraged.

Whole-Genome Sequencing Reveals Diversity of Carbapenem-Resistant Pseudomonas aeruginosa Collected through CDC's Emerging Infections Program, United States, 2016-2018.

The CDC's Emerging Infections Program (EIP) conducted population- and laboratory-based surveillance of US carbapenem-resistant Pseudomonas aeruginosa (CRPA) from 2016 through 2018. To characterize the pathotype, 1,019 isolates collected through this project underwent antimicrobial susceptibility testing and whole-genome sequencing. Sequenced genomes were classified using the seven-gene multilocus sequence typing (MLST) scheme and a core genome (cg)MLST scheme was used to determine phylogeny. Both chromosomal and horizontally transmitted mechanisms of carbapenem resistance were assessed. There were 336 sequence types (STs) among the 1,019 sequenced genomes, and the genomes varied by an average of 84.7% of the cgMLST alleles used. Mutations associated with dysfunction of the porin OprD were found in 888 (87.1%) of the genomes and were correlated with carbapenem resistance, and a machine learning model incorporating hundreds of genetic variations among the chromosomal mechanisms of resistance was able to classify resistant genomes. While only 7 (0.1%) isolates harbored carbapenemase genes, 66 (6.5%) had acquired non-carbapenemase ß-lactamase genes, and these were more likely to have OprD dysfunction and be resistant to all carbapenems tested. The genetic diversity demonstrates that the pathotype includes a variety of strains, and clones previously identified as high-risk make up only a minority of CRPA strains in the United States. The increased carbapenem resistance in isolates with acquired non-carbapenemase ß-lactamase genes suggests that horizontally transmitted mechanisms aside from carbapenemases themselves may be important drivers of the spread of carbapenem resistance in P. aeruginosa.

Using Wolbachia to Eliminate Dengue: Will the Virus Fight Back?

Recent field trials have demonstrated that dengue incidence can be substantially reduced by introgressing strains of the endosymbiotic bacterium Wolbachia into Aedes aegypti mosquito populations. This strategy relies on Wolbachia reducing the susceptibility of Ae. aegypti to disseminated infection by positive-sense RNA viruses like dengue. However, RNA viruses are well known to adapt to antiviral pressures. Here, we review the viral infection stages where selection for Wolbachia-resistant virus variants could occur. We also consider the genetic constraints imposed on viruses that alternate between vertebrate and invertebrate hosts, and the likely selection pressures to which dengue virus might adapt in order to be effectively transmitted by Ae. aegypti that carry Wolbachia. While there are hurdles to dengue viruses developing resistance to Wolbachia, we suggest that long-term surveillance for resistant viruses should be an integral component of Wolbachia-introgression biocontrol programs.

Resistance response to Arenicin derivatives in Escherichia coli.

The rising prevalence of antibiotic resistance poses the greatest health threats. Antimicrobial peptides (AMPs) are regarded as the potentially effective therapy. To avoid current crisis of antibiotic resistance, a comprehensive understanding of AMP resistance is necessary before clinical application. In this study, the development of resistance to the anti-Gram-negative bacteria peptide N6NH2 (21 residues, ß-sheet) was characterized in E. coli ATCC25922. Three N6NH2-resistant E. coli mutants with 32-fold increase in MIC were isolated by serially passaging bacterial lineages in progressively increasing concentrations of N6NH2 and we mainly focus on the phenotype of N6NH2-resistant bacteria different from sensitive bacteria. The results showed that the resistance mechanism was attributed to synergy effect of multiple mechanisms: (i) increase biofilm formation capacity (3 ~ 4-fold); (ii) weaken the affinity of lipopolysaccharide (LPS) with N6NH2 (3 ~ 8-fold); and (iii) change the cell membrane permeability and potential. Interestingly, a chimeric peptide-G6, also a N6NH2 analog, which keep the same antibacterial activity to both wild-type and resistant clones (MIC value: 16 µg/mL), could curb N6NH2-resistant mutants by stronger inhibition of biofilm formation, stronger affinity with LPS, and stronger membrane permeability and depolarization than that of N6NH2.

Molecular Mechanisms of Drug Resistance and Epidemiology of Multidrug-Resistant Variants of Neisseria gonorrhoeae.

The paper presents various issues related to the increasing drug resistance of Neisseria gonorrhoeae and the occurrence and spread of multidrug-resistant clones. One of the most important is the incidence and evolution of resistance mechanisms of N. gonorrhoeae to beta-lactam antibiotics. Chromosomal resistance to penicillins and oxyimino-cephalosporins and plasmid resistance to penicillins are discussed. Chromosomal resistance is associated with the presence of mutations in the PBP2 protein, containing mosaic variants and nonmosaic amino acid substitutions in the transpeptidase domain, and their correlation with mutations in the mtrR gene and its promoter regions (the MtrCDE membrane pump repressor) and in several other genes, which together determine reduced sensitivity or resistance to ceftriaxone and cefixime. Plasmid resistance to penicillins results from the production of beta-lactamases. There are different types of beta-lactamases as well as penicillinase plasmids. In addition to resistance to beta-lactam antibiotics, the paper covers the mechanisms and occurrence of resistance to macrolides (azithromycin), fluoroquinolones and some other antibiotics. Moreover, the most important epidemiological types of multidrug-resistant N. gonorrhoeae, prevalent in specific years and regions, are discussed. Epidemiological types are defined as sequence types, clonal complexes and genogroups obtained by various typing systems such as NG-STAR, NG-MAST and MLST. New perspectives on the treatment of N. gonorrhoeae infections are also presented, including new drugs active against multidrug-resistant strains.

Innovative Anti-CD38 and Anti-BCMA Targeted Therapies in Multiple Myeloma: Mechanisms of Action and Resistance.

CD38 and B-cell maturation antigens (BCMAs) are prevalently expressed on neoplastic plasma cells in multiple myeloma (MM), making them ideal therapeutic targets. Anti-CD38 monoclonal antibodies, such as approved daratumumab and isatuximab, are currently the milestone in MM treatment because they induce plasma cell apoptosis and kill through several mechanisms, including antibody-dependent cellular cytotoxicity or phagocytosis. BCMA is considered an excellent target in MM, and three different therapeutic strategies are either already available in clinical practice or under investigation: antibody-drug conjugates, such as belantamab-mafodotin; bispecific T cell engagers; and chimeric antigen receptor-modified T cell therapies. Despite the impressive clinical efficacy of these new strategies in the treatment of newly diagnosed or multi-refractory MM patients, several mechanisms of resistance have already been described, including antigen downregulation, the impairment of antibody-dependent cell cytotoxicity and phagocytosis, T- and natural killer cell senescence, and exhaustion. In this review, we summarize the current knowledge on the mechanisms of action and resistance of anti-CD38 and anti-BCMA agents and their clinical efficacy and safety.

Revisiting Proteasome Inhibitors: Molecular Underpinnings of Their Development, Mechanisms of Resistance and Strategies to Overcome Anti-Cancer Drug Resistance.

Proteasome inhibitors have shown relevant clinical activity in several hematological malignancies, namely in multiple myeloma and mantle cell lymphoma, improving patient outcomes such as survival and quality of life, when compared with other therapies. However, initial response to the therapy is a challenge as most patients show an innate resistance to proteasome inhibitors, and those that respond to the therapy usually develop late relapses suggesting the development of acquired resistance. The mechanisms of resistance to proteasome inhibition are still controversial and scarce in the literature. In this review, we discuss the development of proteasome inhibitors and the mechanisms of innate and acquired resistance to their activity-a major challenge in preclinical and clinical therapeutics. An improved understanding of these mechanisms is crucial to guiding the design of new and more effective drugs to tackle these devastating diseases. In addition, we provide a comprehensive overview of proteasome inhibitors used in combination with other chemotherapeutic agents, as this is a key strategy to combat resistance.

Acquired resistance to cancer immunotherapy: Role of tumor-mediated immunosuppression.

In the tumor microenvironment (TME), tumor cells are constantly evolving to reduce neoantigen generation and the mutational burden to escape the anti-tumor response. This will lower tumor reactivity to the adaptive immune response and give rise to tumor intrinsic factors, such as altered expression of immune regulatory molecules on tumor cells. Tumor-extrinsic factors, such as immunosuppressive cells, soluble suppressive molecules or inhibitory receptors expressed by immune cells will alter the composition and activity of tumor-infiltrating lymphocytes (TILs) (by increasing T regulatory cells:T effector cells ratio and inhibiting T effector cell function) and promote tumor growth and metastasis. Together, these factors limit the response rates and clinical outcomes to a particular cancer therapy. Within the TME, the cross-talks between immune and non-immune cells result in the generation of positive feedback loops, which augment immunosuppression and support tumor growth and survival (termed as tumor-mediated immunosuppression). Cancer immunotherapies, such as immune checkpoint inhibitors (ICIs) and adoptive cell transfer (ACT), have shown therapeutic efficacy in hematologic cancers and different types of solid tumors. However, achieving durable response rates in some cancer patients remains a challenge as a result of acquired resistance and tumor immune evasion. This could be driven by the cellular and molecular suppressive network within the TME or due to the loss of tumor antigens. In this review, we describe the contribution of the immunosuppressive cellular and molecular tumor network to the development of acquired resistance against cancer immunotherapies. We also discuss potential combined therapeutic strategies which could help to overcome such resistance against cancer immunotherapies, and to enhance anti-tumor immune responses and improve clinical outcomes in patients.

Kinetic and Structural Characterization of the First B3 Metallo-ß-Lactamase with an Active-Site Glutamic Acid.

The structural diversity in metallo-ß-lactamases (MBLs), especially in the vicinity of the active site, has been a major hurdle in the development of clinically effective inhibitors. Representatives from three variants of the B3 MBL subclass, containing either the canonical HHH/DHH active-site motif (present in the majority of MBLs in this subclass) or the QHH/DHH (B3-Q) or HRH/DQK (B3-RQK) variations, were reported previously. Here, we describe the structure and kinetic properties of the first example (SIE-1) of a fourth variant containing the EHH/DHH active-site motif (B3-E). SIE-1 was identified in the hexachlorocyclohexane-degrading bacterium Sphingobium indicum, and kinetic analyses demonstrate that although it is active against a wide range of antibiotics, its efficiency is lower than that of other B3 MBLs but has increased efficiency toward cephalosporins relative to other ß-lactam substrates. The overall fold of SIE-1 is characteristic of the MBLs; the notable variation is observed in the Zn1 site due to the replacement of the canonical His116 by a glutamate. The unusual preference of SIE-1 for cephalosporins and its occurrence in a widespread environmental organism suggest the scope for increased MBL-mediated ß-lactam resistance. Thus, it is relevant to include SIE-1 in MBL inhibitor design studies to widen the therapeutic scope of much needed antiresistance drugs.

Evaluation of FASTinov Ultrarapid Flow Cytometry Antimicrobial Susceptibility Testing Directly from Positive Blood Cultures.

The FASTinov flow cytometry kit, an ultrarapid antimicrobial susceptibility test, was directly evaluated on positive blood cultures (BC) at two sites: (i) FASTinov, S.A., in Porto, Portugal, using BC spiked with well-characterized bacteria, and (ii) Ramón y Cajal University Hospital in Madrid, Spain, using positive BC from patients. Two kits were evaluated, FASTgramneg (Enterobacterales, Pseudomonas, Acinetobacter) and FASTgrampos (Staphylococcus, Enterococcus). Dedicated software for cytometric data analysis and interpretative reporting, including both CLSI and EUCAST criteria, was used. The FASTgramneg kit also provides information about the presence of resistant mechanisms, including extended-spectrum beta-lactamases (ESBLs) and carbapenemases. After 1 h of incubation at 37°C, bacteria were analyzed using a CytoFLEX cytometer (Beckman, CA). Disk diffusion was performed as the reference susceptibility method. Overall, 447 positive BC were included, 100 from hospitalized patients. Categorical agreement values for the FASTgramneg panel were 96.8% based on EUCAST criteria and 96.4% based on CLSI criteria. For the FASTgrampos panel, categorical agreement was 98.6% when using both criteria. When EUCAST criteria were used, the percentages of errors for the FASTgramneg panel were 2.1% minor errors (mE), 1.3% major errors (ME), and 0.6% very major errors (VME). When CLSI criteria were used, 2.9% mE, 0.9% ME, and 0.4% VME were found. VME were mainly observed with amoxicillin-clavulanate, cefotaxime, ceftazidime, and gentamicin. The FASTgrampos panel showed 0.3% mE, 1.4% ME, and 0.4% VME when EUCAST criteria were used (VME with respect to gentamicin and Staphylococcus) and 0.4% mE, 1.4% ME, and no VME when CLSI criteria were used. The FASTinov flow cytometry kits represent a rapid alternative for direct antimicrobial susceptibility testing from positive BC, showing time to results of <2 h, and can be used to personalize antibiotic and stewardship practices.

Molecular epidemiology of aspergillosis in Magellanic penguins and susceptibility patterns of clinical isolates.

Aspergillus section Fumigati is reported in up to 99% of aspergillosis cases in penguins. So far, no data regarding molecular epidemiology and azole resistance are available for A. fumigatus isolates collected from Magellanic penguins. The aim of this work was to perform molecular identification of Aspergillus section Fumigati at species level, to genotype those isolates using microsatellite markers, to evaluate the in vitro susceptibility patterns of A. fumigatus sensu stricto, and to characterize the cyp51A gene in clinical A. fumigatus strains isolated from Magellanic penguins with proven aspergillosis. All 34 isolates included in the study were identified as A. fumigatus sensu stricto. Analyzing the genetic diversity of the isolates of A. fumigatus sensu stricto, we identified two possible outbreaks in the rehabilitation center and we also observed the maintenance of clonal strains through the years. One A. fumigatus sensu stricto isolate was resistant to posaconazole, but the mutations found in the cyp51A gene of this isolate have not been described as conferring phenotypic resistance, suggesting that other mechanisms of resistance could be involved in the resistance of this isolate. With this study, we were able to understand the molecular diversity of Aspergillus fumigatus isolates collected from Magellanic penguins, to characterize them and to associate them with the described global population of Aspergillus fumigatus.

A. fumigatus sensu stricto is of great importance in penguins' aspergillosis. We could identify two outbreaks in the rehabilitation center and the maintenance of clonal strains through the years. Regarding antifungal prophylaxis, it may proceed, but preferably with surveillance for azole resistance.

Current Status of CAR T Cell Therapy for Leukemias.

OPINION STATEMENT: Chimeric antigen receptor (CAR) T-cell therapy has become the standard of care for children and young adults with relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL), and it is a highly promising therapy under investigation for adults with relapsed disease. Despite having potentially life-threatening toxicities, such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, the benefits of CAR T-cell therapy far outweigh these risks, particularly as increased experience and improved supportive care measures are mitigating these toxicities. CAR T cells can result in complete remission for significant proportion of patients with relapsed and refractory B-ALL and permit them to proceed to potentially curative allogeneic hematopoietic stem cell transplantation (allo-HSCT). CAR T cells may also be curative by themselves. Herein lie the greatest challenges and questions for clinical investigators, specifically, how are CAR T cells best employed and how do we overcome mechanisms of resistance to them? The primary clinical question is the timing and even the necessity of allo-HSCT. Relative to resistance, we know that target antigen loss, specifically CD19, is a major contributor to resistance. However, current investigations of alternative targets, such CD22, and CAR T cells expressing dual targeting antigen receptors have demonstrated encouraging initial results and provide a high degree of optimism that the efficacy and the broader application of CAR T-cell therapy will gradually increase in B-ALL. That optimism is not as high and the challenges are increased for the application of CAR T cells in T-cell leukemias and acute myeloid leukemia due to the relative lack of suitable leukemia surface targets that are not also expressed on normal hematopoietic progenitors. Despite these significant challenges, considerable research is being conducted into the development of CAR T cells for these diseases utilizing unique technologies, which may be applicable to other diseases.

Novel Functions and Signaling Specificity for the GraS Sensor Kinase of Staphylococcus aureus in Response to Acidic pH.

Although the GraS sensor kinase of Staphylococcus aureus is known for the sensing of and resistance to cationic antimicrobial peptides (CAMPs), we recently established that it also signals in response to acidic pH, which is encountered on human skin concurrently with CAMPs, antimicrobial unsaturated free fatty acids (uFFA), and calcium. We therefore evaluated how these environmental signals would affect GraS function and resistance to antimicrobial uFFA. Growth at pH 5.5 promoted increased resistance of S. aureus USA300 to linoleic and arachidonic acids but not palmitoleic or sapienic acid. However, enhanced resistance to these C16:1 uFFA was achieved by supplementing acidic medium with 0.5 mM calcium or subinhibitory CAMPs. Enhanced resistance to uFFA at acidic pH was dependent on GraS and GraS-dependent expression of the lysyl-phosphatidylglycerol synthase enzyme MprF, through a mechanism that did not require the lysyl-transferase function of MprF. In addition to enhanced resistance to antimicrobial uFFA, acidic pH also promoted increased production of secreted proteases in a GraS-dependent manner. During growth at pH 5.5, downstream phenotypes of signaling through GraS, including resistance to uFFA, MprF-dependent addition of positive charge to the cell surface, and increased production of secreted proteases, all occurred independently of acidic amino acids in the extracytoplasmic sensor loop of GraS that were previously found to be required for sensing of CAMPs. Cumulatively, our data indicate that signaling through GraS at acidic pH occurs through a mechanism that is distinct from that described for CAMPs, leading to increased resistance to antimicrobial uFFA and production of secreted proteases.IMPORTANCEStaphylococcus aureus asymptomatically colonizes 30% of humans but is also a leading cause of infectious morbidity and mortality. Since infections are typically initiated by the same strain associated with asymptomatic colonization of the nose or skin, it is important to understand how the microbe can endure exposure to harsh conditions that successfully restrict the growth of other bacteria, including a combination of acidic pH, antimicrobial peptides, and antimicrobial fatty acids. The significance of our research is in showing that acidic pH combined with antimicrobial peptide or environmental calcium can signal through a single membrane sensor protein to promote traits that may aid in survival, including modification of cell surface properties, increased resistance to antimicrobial fatty acids, and enhanced production of secreted proteases.

Mapping the Efficacy and Mode of Action of Ethylzingerone [4-(3-Ethoxy-4-Hydroxyphenyl) Butan-2-One] as an Active Agent against Burkholderia Bacteria.

Burkholderia cepacia complex (Bcc) bacteria are intrinsically antimicrobial-resistant opportunistic pathogens and key risk species in the contamination of nonfood industrial products. New agents and formulations to prevent growth of Burkholderia in home care (cleaning agents) and personal-care (cosmetics and toiletries) products are required. We characterized how ethylzingerone [4-(3-ethoxy-4-hydroxyphenyl) butan-2-one] (HEPB) acts as a preservative with activity against Burkholderia species encountered in industry. Burkholderia (n = 58) and non-Burkholderia (n = 7) bacteria were screened for susceptibility to HEPB, and its mode of action and resistance were determined for a model Burkholderia vietnamiensis strain using transposon mutagenesis, transcriptomics, and genome resequencing analysis. The susceptibility of Burkholderia spp. to HEPB (MIC = 0.45% ± 0.11% [wt/vol]; MBC = 0.90% ± 0.3% [wt/vol]) was characterized, with limited inter- and intraspecies differences. HEPB (1% [wt/vol]) was rapidly bactericidal, producing a 6-log reduction in viability within 4 h. Spontaneous resistance to HEPB did not develop, but transient phenotypes with altered growth characteristics and susceptibility to antibiotics were identified after prolonged exposure to sublethal HEPB concentrations. Transposon mutagenesis and RNA-sequencing analysis identified multiple genetic pathways associated with HEPB exposure, including stress response mechanisms, altered permeability, regulation of intracellular pH, damage and repair of intracellular components, and alteration and repair of lipopolysaccharides. Key pathways included the stringent response, homeostasis of intracellular pH by the kdp operon, protection against electrophiles by KefC, and repair of oxidized proteins by methionine sulfoxide reductase enzymes. In summary, we show that HEPB has potent, targeted efficacy against Burkholderia bacteria without promoting wider stable antimicrobial resistance. The mode of action of HEPB against Burkholderia is multifactorial, but killing by intracellular oxidation is a key mechanism of this promising agent.IMPORTANCEBurkholderia bacteria are opportunistic pathogens that can overcome preservatives used in the manufacture of nonsterile industrial products and occasionally cause contamination. Consequently, new preservatives to prevent the growth of key risk Burkholderia cepacia complex bacteria in nonfood industrial products are urgently required. Here, we show that ethylzingerone is active against these problematic bacteria, killing them via a multifactorial mode of action which involves intracellular oxidation.