Evolution of copper coupons resistance in Enterobacteriaceae strains isolated from high-contact surfaces in hospital.

Objective: Metallic copper alloys have gained attention recently as a cutting-edge antibacterial weapon for areas where surface hygiene is crucial. The present study aimed to assess copper coupons (99% Cu) for their potential to decrease the viability of various Enterobacteriaceae strains from inanimate hospital surfaces. Methods: This in vitro-experimental study was conducted at the Microbiology Laboratory, Faculty of Natural and Life Sciences and Earth and Universe Sciences, University of Guelma, and Khodja Ahmed Public Hospital Establishment, Algeria, for a period of six months from January to May 2022. A total of 85 samples were collected from patient room door handles and bed rails at the government hospital in Guelma State, from which 12 enterobacterial isolates were obtained. These isolates were evaluated for susceptibility to copper and polyvinyl chloride (PVC) coupons using plate counts to determine bacterial viability after 72 hours of incubation at 37°C or room temperature (25°C). Antibiotic sensitivity testing was then carried out using a modified Kirby-Bauer disc diffusion method. Copper coupons' ability to either select for or create antibiotic resistance is also determined. Results: Copper showed a bactericidal effect after three hours for Serratia odorifera and six hours for Escherichia coli. Whereas it was shown that within three days of selection, 83.33% of Enterobacteriaceae strains are capable of rapidly acquiring Cu resistance. Indeed, the increase in temperature reduced the effects of Cu (p<0.05; Student's t-test). Antimicrobial susceptibility testing revealed that the copper-resistant bacteria were less sensitive than their predecessors. Citrobacter freundii strains showed the highest incidence of multidrug resistance. The most significant findings included widespread resistance to beta-lactams (100%-75%) and chloramphenicol (66.67%). Conclusion: These results suggest that prolonged copper usage may contribute to the development of antibiotic resistance, which could have significant ramifications.

Structural variations and roles of rhamnose-rich cell wall polysaccharides in Gram-positive bacteria.

Rhamnose-rich cell wall polysaccharides (Rha-CWPSs) have emerged as crucial cell wall components of numerous Gram-positive, ovoid-shaped bacteria-including streptococci, enterococci, and lactococci-of which many are of clinical or biotechnological importance. Rha-CWPS are composed of a conserved polyrhamnose backbone with side-chain substituents of variable size and structure. Because these substituents contain phosphate groups, Rha-CWPS can also be classified as polyanionic glycopolymers, similar to wall teichoic acids, of which they appear to be functional homologs. Recent advances have highlighted the critical role of these side-chain substituents in bacterial cell growth and division, as well as in specific interactions between bacteria and infecting bacteriophages or eukaryotic hosts. Here, we review the current state of knowledge on the structure and biosynthesis of Rha-CWPS in several ovoid-shaped bacterial species. We emphasize the role played by multicomponent transmembrane glycosylation systems in the addition of side-chain substituents of various sizes as extracytoplasmic modifications of the polyrhamnose backbone. We provide an overview of the contribution of Rha-CWPS to cell wall architecture and biogenesis and discuss current hypotheses regarding their importance in the cell division process. Finally, we sum up the critical roles that Rha-CWPS can play as bacteriophage receptors or in escaping host defenses, roles that are mediated mainly through their side-chain substituents. From an applied perspective, increased knowledge of Rha-CWPS can lead to advancements in strategies for preventing phage infection of lactococci and streptococci in food fermentation and for combating pathogenic streptococci and enterococci.

Urgent, Comprehensive Federal Action Needed To Stem Mortality and Medicare Costs Associated With Antimicrobial Resistance.

This paper is a call to action for the policies necessary to reduce the burden of antimicrobial resistance, including federal investments in antibiotic stewardship, antibiotic innovation, surveillance, research, diagnostics, infection prevention, the infectious diseases workforce, and global coordination.

RNase HI Depletion Strongly Potentiates Cell Killing by Rifampicin in Mycobacteria.

Multidrug-resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and they can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are nonviable. This essentiality makes it a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin, and rifampicin was increased, the latter by a striking near 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and rifampicin. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action and providing the first step in developing a new class of antimycobacterial drug.

A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier.

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the ß-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes.

Bacteria account for 1000-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different peptidoglycan-based cell wall structures that encase bacterial cells. Although the basic structure of peptidoglycan is highly conserved, consisting of long glycan strands that are cross-linked by short peptide chains, the mature cell wall is chemically diverse. Peptidoglycan hydrolases and cell wall-tailoring enzymes that regulate glycan strand length, the degree of cross-linking, and the addition of other modifications to peptidoglycan are central in determining the final architecture of the bacterial cell wall. Historically, it has been difficult to biochemically characterize these enzymes that act on peptidoglycan because suitable peptidoglycan substrates were inaccessible. In this review, we discuss fundamental aspects of bacterial cell wall synthesis, describe the regulation and diverse biochemical and functional activities of peptidoglycan hydrolases, and highlight recently developed methods to make and label defined peptidoglycan substrates. We also review how access to these substrates has now enabled biochemical studies that deepen our understanding of how bacterial cell wall enzymes cooperate to build a mature cell wall. Such improved understanding is critical to the development of new antibiotics that disrupt cell wall biogenesis, a process essential to the survival of bacteria.

Assessing Animal Models of Bacterial Pneumonia Used in Investigational New Drug Applications for the Treatment of Bacterial Pneumonia.

Animal models of bacterial infection have been widely used to explore the in vivo activity of antibacterial drugs. These data are often submitted to the U.S. Food and Drug Administration to support human use in an investigational new drug application (IND). To better understand the range and scientific use of animal models in regulatory submissions, a database was created surveying recent pneumonia models submitted as part of IND application packages. The IND studies were compared to animal models of bacterial pneumonia published in the scientific literature over the same period of time. In this review, we analyze the key experimental design elements, such as animal species, immune status, pathogens selected, and route of administration, and study endpoints.

White Paper: Developing Antimicrobial Drugs for Resistant Pathogens, Narrow-Spectrum Indications, and Unmet Needs.

Despite progress in antimicrobial drug development, a critical need persists for new, feasible pathways to develop antibacterial agents to treat people infected with drug-resistant bacteria. Infections due to resistant gram-negative bacilli continue to cause unacceptable morbidity and mortality rates. Antibacterial agents have been historically studied in noninferiority clinical trials that focus on a single site of infection (eg, complicated urinary tract infections, intra-abdominal infections), yet these designs may not be optimal, and often are not feasible, for study of infections caused by drug-resistant bacteria. Over the past several years, multiple stakeholders have worked to develop consensus regarding paths forward with a goal of facilitating timely conduct of antimicrobial development. Here we advocate for a novel and pragmatic approach and, toward this end, present feasible trial designs for antibacterial agents that could enable conduct of narrow-spectrum, organism-specific clinical trials and ultimately approval of critically needed new antibacterial agents.

Efficient Delivery of Investigational Antibacterial Agents via Sustainable Clinical Trial Networks.

The economics of antibiotics can be improved by infectious diseases-specific clinical trial networks. While developers would still need to implement an independent phase 1 program as well as studies focused on highly resistant pathogens, standardized procedures in a network focused on usual drug resistance phenotype isolates would permit sharing of controls and would predictably generate high-quality pivotal data for product registration while creating cost and time savings in the range of 30%-40%. This would reduce economic barriers to antibiotic development and contribute to public health.

Persisters-as elusive as ever.

Persisters-a drug-tolerant sub-population in an isogenic bacterial culture-have been featured throughout the last decade due to their important role in recurrent bacterial infections. Numerous investigations detail the mechanisms responsible for the formation of persisters and suggest exciting strategies for their eradication. In this review, we argue that the very term "persistence" is currently used to describe a large and heterogeneous set of physiological phenomena that are functions of bacterial species, strains, growth conditions, and antibiotics used in the experiments. We caution against the oversimplification of the mechanisms of persistence and urge for a more rigorous validation of the applicability of these mechanisms in each case.

A critical analysis of the review on antimicrobial resistance report and the infectious disease financing facility.

Over the past year, two major policy initiatives have been introduced focusing on stimulating antibiotic development for human consumption. The European Investment Bank has announced the development of the Infectious Disease Financing Facility (IDFF) and the British government commissioned the Review on Antimicrobial Resistance, led by Jim O'Neill. Each constitutes a major effort by the European community to address the evolving crisis of antimicrobial resistance. Though both have similar goals, the approaches are unique and worthy of consideration.This manuscript utilizes a previously published framework for evaluation of antibiotic incentive plans to clearly identify the strengths and weaknesses of each proposal. The merits of each proposal are evaluated in how they satisfy four key objectives: 1) Improve the overall net present value (NPV) for new antibiotic projects; 2) Enable greater participation of Small to Medium Sized Enterprises (SME); 3) Encourage participation by large pharmaceutical companies; 4) Facilitate cooperation and synergy across the antibiotic market. The IDFF seeks to make forgivable loans to corporations with promising compounds, while the O'Neill group proposes a more comprehensive framework of early stage funding, along with the creation of a stable global market.Ultimately, the proposals may prove complementary and if implemented together may form a more comprehensive plan to address an impending global crisis. Substantial progress will only be made on these efforts if action is taken at an international level, therefore we recommend consideration of these efforts at the upcoming G20 summit.

Identifying potential therapeutic targets of methicillin-resistant Staphylococcus aureus through in vivo proteomic analysis.

BACKGROUND: Detailed knowledge on protein repertoire of a pathogen during host infection is needed for both developing a better understanding of the pathogenesis and defining potential therapeutic targets. Such data, however, have been missing for Staphylococcus aureus, a major human pathogen. METHODS: We determined the surface proteome of methicillin-resistant S. aureus (MRSA) clone usa300 derived directly from murine systemic infectiON. RESULTS: The majority of the in vivo-expressed surface-associated proteins were lipoproteins involved in nutrient acquisition, especially uptake of metal ions. Enzyme-linked immunosorbent assay (ELISA) of convalescent human serum samples revealed that proteins that were highly produced during murine experimental infection were also produced during natural human infection. We found that among the 7 highly abundant lipoproteins only MntC, which is the manganese-binding protein of the MntABC system, was essential for MRSA virulence during murine systemic infection. Moreover, we show that MntA and MntB are equally important for MRSA virulence. CONCLUSIONS: Besides providing experimental evidence that MntABC might be a potential therapeutic target for the development of antibiotics, our in vivo proteomics data will serve as a valuable basis for defining potential antigen combinations for multicomponent vaccines.

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles.

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

A panel of genotypically and phenotypically diverse clinical Acinetobacter baumannii strains for novel antibiotic development.

Acinetobacter baumannii is one of the most important pathogens worldwide. The intrinsic and acquired resistance of A. baumannii, coupled with the slow pace of novel antimicrobial drug development, poses an unprecedented and enormous challenge to clinical anti-infective therapy of A. baumannii. Recent studies in the field of pathogenicity, antibiotic resistance, and biofilms of A. baumannii have focused on the model strains, including ATCC 17978, ATCC 19606, and AB5075. However, these model strains represent only a limited portion of the heterogeneity in A. baumannii. Furthermore, variants of these model strains have emerged that show significant diversity not only at the genotypic level but also reflected in differences at the phenotypic levels of capsule, virulence, pathogenicity, and antibiotic resistance. Research on A. baumannii, a key pathogen, would benefit from a standardized approach, which characterizes heterogeneous strains in order to facilitate rapid diagnosis, discovery of new therapeutic targets, and efficacy assessment. Our study provides and describes a standardized, genomically and phenotypically heterogeneous panel of 45 different A. baumannii strains for the research community. In addition, we performed comparative analyses of several phenotypes of this panel. We found that the sequence type 2 (ST2) group showed significantly higher rates of resistance, lower fitness cost for adaptation, and yet less biofilm formation. The Macrocolony type E (MTE, flat center and wavy edge phenotype reported in the literature) group showed a less clear correlation of resistance rates and growth rate, but was observed to produce more biofilms. Our study sheds light on the complex interplay of resistance fitness and biofilm formation within distinct strains, offering insights crucial for combating A. baumannii infection. IMPORTANCE: Acinetobacter baumannii is globally notorious, and in an effort to combat the spread of such pathogens, several emerging candidate therapies have already surfaced. However, the strains used to test these therapies vary across studies (the sources and numbers of test strains are varied and often very large, with little heterogeneity). The variation complicates the studies. Furthermore, the limited standardized resources of A. baumannii strains have greatly restricted the research on the physiology, pathogenicity, and antibiotic resistance. Therefore, it is crucial for the research community to acquire a standardized and heterogeneous panel of A. baumannii. Our study meticulously selected 45 diverse A. baumannii strains from a total of 2,197 clinical isolates collected from 64 different hospitals across 27 provinces in China, providing a scientific reference for the research community. This assistance will significantly facilitate scientific exchange in academic research.

Streptococcus mutans inhibits the growth of Enterococcus via the non-ribosomal cyclic peptide mutanobactin.

Enterococcus faecalis is a Gram-positive commensal bacterium in the gastrointestinal tract and an opportunistic pathogen. Enterococci are a leading cause of nosocomial infections, treatment of which is complicated by intrinsic and acquired antibiotic resistance mechanisms. Additionally, E. faecalis has been associated with various oral diseases, and it is frequently implicated in the failure of endodontic treatment. For establishment and persistence in a microbial community, E. faecalis must successfully compete against other bacteria. Streptococcal species play an important role in the establishment of the oral microbiome and co-exist with Enterococcus in the small intestine, yet the nature of interactions between E. faecalis and oral streptococci remains unclear. Here, we describe a mechanism by which Streptococcus mutans inhibits the growth of E. faecalis and other Gram-positive pathogens through the production of mutanobactin, a cyclic lipopeptide. Mutanobactin is produced by a polyketide synthase-nonribosomal peptide synthetase hybrid system encoded by the mub locus. Mutanobactin-producing S. mutans inhibits planktonic and biofilm growth of E. faecalis and is also active against other Enterococcus species and Staphylococcus aureus. Mutanobactin damages the cell envelope of E. faecalis, similar to other lipopeptide antibiotics like daptomycin. E. faecalis resistance to mutanobactin is mediated by the virulence factor gelatinase, a secreted metalloprotease. Our results highlight the anti-biofilm potential of the microbial natural product mutanobactin, provide insight into how E. faecalis interacts with other organisms in the human microbiome, and demonstrate the importance of studying E. faecalis dynamics within polymicrobial communities.

Antibiotic Culture: A History of Antibiotic Use in the Second Half of the 20th and Early 21st Century in the People's Republic of China.

Antimicrobial resistance is now widely regarded as a global public health threat. A growing number of studies suggest that antibiotic resistance is higher in China than in most western countries. Despite the current official regulation prohibiting pharmacies from the unrestricted selling of antibiotics, there is little sign of declining consumer demand. China now ranks as the second largest consumer of antibiotics in the world, after India. Drawing on published historical data, unpublished archival documents, and recently collected oral interviews, this paper provides a historical overview of antibiotic use and abuse in the People's Republic of China (PRC) from the second half of the 20th century to the present. It demonstrates how the political demand for health improvement, along with the state-sponsored popularization of allopathic medicine, on the one hand, and the lack of access to adequate medical care for the majority of the population, as well as the existing culture of self-medication, on the other hand, are working in tandem to create antibiotic dependency in China. In addition, the privatization and marketization of biomedicine and health care in post-Mao China have helped to build a new and ever-thriving network of production, distribution, and marketing of antibiotics, which has often proven difficult for the authorities to monitor. At the same time, increased purchasing power and easier accessibility created by this new network of production, distribution, and marketing have further contributed to the prevalence of antibiotic overuse in the late 20th and early 21st centuries.

Investigating forthcoming strategies to tackle deadly superbugs: current status and future vision.

INTRODUCTION: Superbugs are microorganisms that cause disease and have increased resistance to the treatments typically used against infections. Recently, antibiotic resistance development has been more rapid than the pace at which antibiotics are manufactured, leading to refractory infections. Scientists are concerned that a particularly virulent and lethal 'superbug' will one day join the ranks of existing bacteria that cause incurable diseases, resulting in a global health disaster on the scale of the Black Death. AREAS COVERED: This study highlights the current developments in the management of antibiotic-resistant bacteria and recommends strategies for further regulating antibiotic-resistant microorganisms associated with the healthcare system. This review also addresses the origins, prevalence, and pathogenicity of superbugs, and the design of antibacterial against these growing multidrug-resistant organisms from a medical perspective. EXPERT OPINION: It is recommended that antimicrobial resistance should be addressed by limiting human-to-human transmission of resistant strains, lowering the use of broad-spectrum antibiotics, and developing novel antimicrobials. Using the risk-factor domains framework from this study would assure that not only clinical but also community and hospital-specific factors are covered, lowering the chance of confounders. Extensive subjective research is necessary to fully understand the underlying factors and uncover previously unexplored areas.

A 3-alkylpyridine-bearing alkaloid exhibits potent antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) with no detectable resistance.

Staphylococcus aureus is a Gram-positive bacterium responsible for a wide variety of infectious diseases, and its methicillin-resistant isolates pose a serious worldwide public health risk. New drugs are urgently needed for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we evaluated the antibacterial activity of five 3-alkyl-pyridinic analogs against MRSA and, of these compounds, compound 6 showed promising antibacterial activity against Staphylococcus with minimum inhibitory concentration (MIC) ranging from 0.98 to 3.9 µgmL-¹ . In addition, it exhibited a rapid bactericidal action, with complete elimination of MRSA after 6 h of incubation at 15.6 µgmL-¹ . Compound 6 had the ability to damage the bacterial membrane and induce cell lysis and, due to its action on the membrane, showed low resistance induction potential in vitro. In the combination study, compound 6 revealed an additive effect (FICI = 1) with vancomycin and ofloxacin and ciprofloxacin (FICI = 0.75) against MRSA, reducing the effective concentration of this antibiotic two-fold. The anti-staphylococcal activity of compound 6 was stable in the presence of different concentrations of NaCl (50, 200, and 400 µM), trypsin ( 1:500, 1:250) and under a variety of pH values (4, 5, 6, and 8); however, its binding to plasmatic proteins (i.e., albumin) was substantial. The previous exposure of MRSA to the compound was able to reduce the formation of bacterial biofilm and reduce the biomass of mature biofilms. Compound 6 showed low selectivity in vitro for MRSA USA 300 when compared to eukaryotic cells (epithelial, fibroblast, and red blood cells).