Antibiotics

Honeybee products are a great source of polyphenols with recognized applications in dental medicine. Although their biological mechanisms in oral diseases are not fully understood, numerous in vitro, in vivo and clinical studies have reported promising results in the prevention and treatment of oral diseases. Bioactivities, such as antibacterial, antiviral, antiparasite, anticancer, anti-inflammatory and anti-oxidant properties, recommend their future study in order to develop efficient alternatives in the management of widespread oral conditions, such as dental caries and periodontitis. The most investigated mechanisms of polyphenols in oral health rely on their ability to strengthen the dental enamel, decrease the development of dental plaque formation, inhibit the progression of dental caries and development of dental pathogens and show anti-inflammatory properties. These features recommend them as useful honeybee candidates in the management of emerging oral diseases. Full article

Antibiotic resistance is on the rise and has become one of the biggest public health challenges of our time. Bacteria are able to adapt to the selective pressure exerted by antibiotics in numerous ways, including the (over)expression of efflux pumps, which represents an ancient bacterial defense mechanism. Several studies show that overexpression of efflux pumps rarely provides clinical resistance but contributes to a low-level resistance, which allows the bacteria to persist at the infection site. Furthermore, recent studies show that efflux pumps, apart from pumping out toxic substances, are also linked to persister formation and increased spontaneous mutation rates, both of which could aid persistence at the infection site. Surviving at the infection site provides the low-level-resistant population an opportunity to evolve by acquiring secondary mutations in antibiotic target genes, resulting in clinical resistance to the treating antibiotic. Thus, this emphasizes the importance and challenge for clinicians to be able to monitor overexpression of efflux pumps before low-level resistance develops to clinical resistance. One possible treatment option could be an efflux pump-targeted approach using efflux pump inhibitors. Full article

One of the challenges of modern biotechnology is to find new routes to mitigate the resistance to conventional antibiotics. Antimicrobial peptides (AMPs) are an alternative type of biomolecules, naturally present in a wide variety of organisms, with the capacity to overcome the current microorganism resistance threat. Here, we reviewed our recent efforts to develop a new library of non-rationally produced AMPs that relies on bacterial genome inherent diversity and compared it with rationally designed libraries. Our approach is based on a four-stage workflow process that incorporates the interplay of recent developments in four major emerging technologies: artificial intelligence, molecular dynamics, surface-display in microorganisms, and microfluidics. Implementing this framework is challenging because to obtain reliable results, the in silico algorithms to search for candidate AMPs need to overcome issues of the state-of-the-art approaches that limit the possibilities for multi-space data distribution analyses in extremely large databases. We expect to tackle this challenge by using a recently developed classification algorithm based on deep learning models that rely on convolutional layers and gated recurrent units. This will be complemented by carefully tailored molecular dynamics simulations to elucidate specific interactions with lipid bilayers. Candidate AMPs will be recombinantly-expressed on the surface of microorganisms for further screening via different droplet-based microfluidic-based strategies to identify AMPs with the desired lytic abilities. We believe that the proposed approach opens opportunities for searching and screening bioactive peptides for other applications. Full article

The present study surveys potential antibacterial synergism effects of silver nitrate with eight other metal or metalloid-based antimicrobials (MBAs), including silver nitrate, copper (II) sulfate, gallium (III) nitrate, nickel sulfate, hydrogen tetrachloroaurate (III) trihydrate (gold), aluminum sulfate, sodium selenite, potassium tellurite, and zinc sulfate. Bacteriostatic and bactericidal susceptibility testing explored antibacterial synergism potency of 5760 combinations of MBAs against three bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus) in three different media. Silver nitrate in combination with potassium tellurite, zinc sulfate, and tetrachloroaurate trihydrate had remarkable bactericidal and bacteriostatic synergism effects. Synergism properties of MBAs decreased effective antibacterial concentrations remarkably and bacterial cell count decreased by 8.72 log10 colony-forming units (CFU)/mL in E. coli, 9.8 log10 CFU/mL in S. aureus, and 12.3 log10 CFU/mL in P. aeruginosa, compared to each MBA alone. Furthermore, most of the MBA combinations inhibited the recovery of bacteria; for instance, the combination of silver nitrate–tetrachloroaurate against P. aeruginosa inhibited the recovery of bacteria, while three-fold higher concentration of silver nitrate and two-fold higher concentration of tetrachloroaurate were required for inhibition of recovery when used individually. Overall, higher synergism was typically obtained in simulated wound fluid (SWF) rather than laboratory media. Unexpectedly, the combination of A silver nitrate–potassium tellurite had antagonistic bacteriostatic effects in Luria broth (LB) media for all three strains, while the combination of silver nitrate–potassium tellurite had the highest bacteriostatic and bactericidal synergism in SWF. Here, we identify the most effective antibacterial MBAs formulated against each of the Gram-positive and Gram-negative pathogen indicator strains. Full article

This study aimed at the characterization of carbapenem-resistant Klebsiella pneumoniae isolates focusing on typing of the bla_OXA-48-like genes. Additionally, the correlation between the resistance pattern and biofilm formation capacity of the carbapenem-resistant K. pneumoniae isolates was studied. The collected isolates were assessed for their antimicrobial resistance and carbapenemases production by a modified Hodge test and inhibitor-based tests. The carbapenemases encoding genes (bla_KPC, bla_NDM, bla_VIM, bla_IMP, and bla_OXA-48-like) were detected by PCR. Isolates harboring bla_OXA-48-like genes were genotyped by Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) and plasmid profile analysis. The discriminatory power of the three typing methods (antibiogram, ERIC-PCR, and plasmid profile analysis) was compared by calculation of Simpson’s Diversity Index (SDI). The transferability of bla_OXA-48 gene was tested by chemical transformation. The biofilm formation capacity and the prevalence of the genes encoding the fimbrial adhesins (fimH-1 and mrkD) were investigated. The isolates showed remarkable resistance to β-lactams and non-β-lactams antimicrobials. The coexistence of the investigated carbapenemases encoding genes was prevalent except for only 15 isolates. The plasmid profile analysis had the highest discriminatory power (SDI = 0.98) in comparison with ERIC-PCR (SDI = 0.89) and antibiogram (SDI = 0.78). The transferability of bla_OXA-48 gene was unsuccessful. All isolates were biofilm formers with the absence of a significant correlation between the biofilm formation capacity and resistance profile. The genes fimH-1 and mrkD were prevalent among the isolates. The prevalence of carbapenemases encoding genes, especially bla_OXA-48-like genes in Egyptian healthcare settings, is worrisome and necessitates further strict dissemination control measures. Full article

Bloodstream infections (BSIs) are among the leading causes of morbidity and mortality worldwide, among infectious diseases. Local knowledge of the main bacteria involved in BSIs and their associated antibiotic susceptibility patterns is essential to rationalize the empiric antimicrobial therapy. The aim of this study was to define the incidence of infection and evaluate the antimicrobial resistance profile of the main pathogens involved in BSIs. This study enrolled patients of all ages and both sexes admitted to the University Hospital “San Giovanni di Dio e Ruggi d’Aragona”, Salerno, Italy between January 2015 to December 2019. Bacterial identification and antibiotic susceptibility testing were performed with Vitek 2. A number of 3.949 positive blood cultures were included out of 24,694 total blood cultures from 2015 to 2019. Coagulase-negative staphylococci (CoNS) were identified as the main bacteria that caused BSI (17.4%), followed by Staphylococcus aureus (12.3%), Escherichia coli (10.9%), and Klebsiella pneumoniae (9.4%). Gram-positive bacteria were highly resistant to Penicillin G and Oxacillin, while Gram-negative strains to Ciprofloxacin, Cefotaxime, Ceftazidime, and Amoxicillin-clavulanate. High susceptibility to Vancomycin, Linezolid, and Daptomycin was observed among Gram-positive strains. Fosfomycin showed the best performance to treatment Gram-negative BSIs. Our study found an increase in resistance to the latest generation of antibiotics over the years. This suggests an urgent need to improve antimicrobial management programs to optimize empirical therapy in BSI. Full article

The increased use of antibiotics in food animals has resulted in the selection of drug-resistant bacteria across the farm-to-fork continuum. This study aimed to investigate the molecular epidemiology of antibiotic-resistant Escherichia coli from intensively produced poultry in the uMgungundlovu District, KwaZulu-Natal, South Africa. Samples were collected weekly between August and September 2017 from hatching to final retail products. E. coli was isolated on eosin methylene blue agar, identified biochemically, and confirmed using polymerase chain reaction (PCR). Susceptibility to 19 antibiotics was ascertained by the Kirby–Bauer disc diffusion method. PCR was used to test for resistance genes. The clonal similarity was investigated using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). In total, 266 E. coli isolates were obtained from all the samples, with 67.3% being non-susceptible to at least one antibiotic tested and 6.7% multidrug resistant. The highest non-susceptibility was to ampicillin (48.1%) and the lowest non-susceptibility to ceftriaxone and azithromycin (0.8%). Significant non-susceptibility was observed to tetracycline (27.4%), nalidixic acid (20.3%), trimethoprim-sulfamethoxazole (13.9%), and chloramphenicol (11.7%) which have homologues used in the poultry industry. The most frequently observed resistance genes were bla_CTX-M (100%), sul1 (80%), tetA (77%), and tetB (71%). ERIC-PCR grouped isolates into 27 clusters suggesting the spread of diverse clones across the farm-to-fork continuum. This reiterates the role of intensive poultry farming as a reservoir and a potential vehicle for the transmission of antibiotic resistance, with potentially severe public health implications, thus, requiring prompt and careful mitigation measures to protect human and environmental health. Full article

Probiotics have been shown to bind to host receptors, which are important for pathogen adhesion and induce the host’s production of defence factors. They can activate the goblet-cell-derived production of mucins, a major component of the mucus layer and a physical barrier participating in limiting the proximity of microorganisms to the epithelial layer. In the last decade, Bacillus spp. strains have gained interest in human and animal health due to their tolerance and stability under gastrointestinal tract conditions. Moreover, Bacillus spp. strains can also produce various antimicrobial peptides that can support their use as commercial probiotic supplements and functional foods. The present study aimed to evaluate and determine the ability of selected Bacillus spp. strains to inhibit the growth of enterotoxigenic Escherichia coli (ETEC) F4 and to reduce binding of ETEC F4 to HT29-16E (mucus-secreting and goblet-like) human intestinal cells. Moreover, mucus production in the HT29 cells in the presence of the Bacillus spp. strains was quantified by ELISA. Bacillus spp. strains (CHCC 15076, CHCC 15516, CHCC 15541, and CHCC 16872) significantly inhibited the growth of ETEC F4. Moreover, the ability of the probiotic Bacillus spp. strains to stimulate mucin release was highly strain dependent. The treatment with Bacillus subtilis CHCC 15541 resulted in a significant increase of both MUC2 and MUC3 in HT29-16E cells. Therefore, this strain could be an up-and-coming candidate for developing commercial probiotic supplements to prevent infections caused by ETEC F4 and, potentially, other pathogens. Full article

Multiple modes of interventions are available when implementing an antibiotic stewardship program (ASP), however, their complementarity has not yet been assessed. In a 938-bed hospital, we sequentially implemented four combined modes of interventions over one year, centralized by one infectious diseases specialist (IDS): (1) on-request infectious diseases specialist consulting service (IDSCS), (2) participation in intensive care unit meetings, (3) IDS intervention triggered by microbiological laboratory meetings, and (4) IDS intervention triggered by pharmacist alert. We assessed the complementarity of the different cumulative actions through quantitative and qualitative analysis of all interventions traced in the electronic medical record. We observed a quantitative and qualitative complementarity between interventions directly correlating to a decrease in antibiotic use. Quantitatively, the number of interventions has doubled after implementation of IDS intervention triggered by pharmacist alert. Qualitatively, these kinds of interventions led mainly to de-escalation or stopping of antibiotic therapy (63%) as opposed to on-request IDSCS (32%). An overall decrease of 14.6% in antibiotic use was observed (p = 0.03). Progressive implementation of the different interventions showed a concrete complementarity of these actions. Combined actions in ASPs could lead to a significant decrease in antibiotic use, especially regarding critical antibiotic prescriptions, while being well accepted by prescribers. Full article

Bioactive natural products are typically secreted by the producer strain. Besides that, this allows the targeting of competitors, also filling a protective role, reducing the chance of self-killing. Surprisingly, DNA-degrading and membrane damaging prodiginines (PdGs) are only produced intracellularly, and are required for the onset of the second round of programmed cell death (PCD) in Streptomyces coelicolor. In this work, we investigated the influence of PdGs on the timing of the morphological differentiation of S. coelicolor. The deletion of the transcriptional activator gene redD that activates the red cluster for PdGs or nutrient-mediated reduction of PdG synthesis both resulted in the precocious appearance of mature spore chains. Transcriptional analysis revealed an accelerated expression of key developmental genes in the redD null mutant, including bldN for the developmental σ factor BldN which is essential for aerial mycelium formation. In contrast, PdG overproduction due to the enhanced copy number of redD resulted in a delay or block in sporulation. In addition, confocal fluorescence microscopy revealed that the earliest aerial hyphae do not produce PdGs. This suggests that filaments that eventually differentiate into spore chains and are hence required for survival of the colony, are excluded from the second round of PCD induced by PdGs. We propose that one of the roles of PdGs would be to delay the entrance of S. coelicolor into the dormancy state (sporulation) by inducing the leakage of the intracellular content of dying filaments thereby providing nutrients for the survivors. Full article

The inhibition of a bacterial cell division protein, filamentous temperature-sensitive Z (FtsZ), prevents the reproduction of Mycobacteria. To propose potent inhibitors of FtsZ, the binding properties of FtsZ with various derivatives of Zantrin ZZ3 were investigated at an electronic level, using molecular simulations. We here employed protein–ligand docking, classical molecular mechanics (MM) optimizations, and ab initio fragment molecular orbital (FMO) calculations. Based on the specific interactions between FtsZ and the derivatives, as determined by FMO calculations, we proposed novel ligands, which can strongly bind to FtsZ and inhibit its aggregations. The introduction of a hydroxyl group into ZZ3 was found to enhance its binding affinity to FtsZ. Full article

The negligible water solubility of tetracycline (TC), a well-known antibiotic of clinical use, is the major disadvantage for its oral administration. With the aim to improve the water solubility of TC, the micelles of formulae [email protected] and [email protected] (SLS = sodium lauryl sulphate and CTAB = cetrimonium bromide) were synthesized. The micelles [email protected] and [email protected] were characterized by melting point (m.p.), thermogravimetric differential thermal analysis (TG-DTA), differential scanning calorimetry (DTG/DSC), attenuated total reflection spectroscopy (FT-IR-ATR), ultra-violet visible (UV/vis) spectroscopy, proton nucleus magnetic resonance (¹H-NMR) spectroscopy, and the ultrasonically-induced biregringence technique. The antimicrobial activity of [email protected] and [email protected] was evaluated, by means of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and inhibition zone (IZ), against the Gram negative bacterial strains Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) and the Gram positive ones of the genus of Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus). Generally, both micelles show better activity than that of TC against the microbial strains tested. Thus, the MIC value of [email protected] is 550-fold higher than that of free TC against S. epidermidis. Despite the stronger activity of [email protected] than [email protected] against both Gram negative and Gram positive microbes, [email protected] is classified as a bactericidal agent (in that it eliminates 99.9% of the microbes), in contrast to [email protected], which is bacteriostatic one (inhibits, but does not kill the organisms). The toxicity of [email protected] and [email protected] was evaluated against human corneal eukaryotic cells (HCECs). Moreover, [email protected] and [email protected] exhibit low in vivo toxicity against Artemia salina, even at concentrations up to threefold higher than those of their MIC^max. Therefore, [email protected] and [email protected] can be candidates for the development of new antibiotics. Full article

The increasing number of infections caused by multidrug-resistant bacteria requires an intensified search for new antibiotics. Pep19-4LF is a synthetic antimicrobial peptide (GKKYRRFRWKFKGKLFLFG) that was previously designed with the main focus on high antimicrobial activity. The hydrophobic motif, LFLFG, was found to be essential for antimicrobial activity. However, this motif shows several limitations such as aggregation in biological media, low solubility, and small yields in peptide synthesis. In order to obtain more appropriate peptide characteristics, the hydrophobic motif was replaced with fatty acids. For this purpose, a shortened variant of Pep19-4LF (Pep19-short; GKKYRRFRWKFKGK) was synthesized and covalently linked to saturated fatty acids of different chain lengths. The peptide conjugates were tested with respect to their antibacterial activity by microdilution experiments on different bacterial strains. The length of the fatty acid was found to be directly correlated to the antimicrobial activity up to an ideal chain length (undecanoic acid, C11:0). This conjugate showed high antimicrobial activity in absence of toxicity. Time–kill studies revealed a fast and bactericidal mode of action. Furthermore, the first in vivo experiments of the conjugate in rodents demonstrated pharmacokinetics appropriate for application as a drug. These results clearly indicate that the hydrophobic motif of the peptide can be replaced by a single fatty acid of medium length, simplifying the design of this antimicrobial peptide while retaining high antimicrobial activity in the absence of toxicity. Full article

Species of the family Bufonidae, better known as true toads, are widespread and produce bioactive substances in the secretions obtained from specialized skin macroglands. Some true toads have been employed as a folk remedy to treat infectious diseases caused by microbial pathogens. Recent publications based on in silico analysis highlighted the Bufonidae as promising sources of antimicrobial peptides. A review of the literature reveals that Bufonidae skin secretion extracts show inhibitory activity in vitro against clinical isolates of bacteria, resistant and standard strains of bacterial, and fungal and parasitic human pathogens. Secondary metabolites belonging to the classes of alkaloids, bufadienolides, and peptides with antimicrobial activity have been isolated from species of the genera Bufo, Bufotes, Duttaphrynus, and Rhinella. Additionally, some antimicrobial extracts and purified compounds display low cytotoxicity against mammal cells. Full article

Agricultural fungicides contaminate the environment and promote the spread of fungicide-resistant strains of pathogenic fungi. The enhancement of pathogen sensitivity to these pesticides using chemosensitizers allows the reducing of fungicide dosages without a decrease in their efficiency. Using Petri plate and microplate bioassays, 6-demethylmevinolin (6-DMM), a putative sensitizer of a microbial origin, was shown to affect both colony growth and conidial germination of Alternaria solani, A. alternata, Parastagonospora nodorum, Rhizoctonia solani, and four Fusarium species (F. avenaceum, F. culmorum, F. oxysporum, F. graminearum) forming a wheat root rot complex together with B. sorokiniana. Non- or marginally toxic 6-DMM concentrations suitable for sensitizing effect were determined by the probit analysis. The range of determined concentrations confirmed a possibility of using 6-DMM as a putative sensitizer for the whole complex of root rot agents, other cereal pathogens (A. alternata, P.nodorum), and some potato (R. solani, A. solani) and tomato (A. solani) pathogens. Despite the different sensitivities of the eight tested pathogens, 6-DMM lacked specificity to fungi and possessed a mild antimycotic activity that is typical of other known pathogen-sensitizing agents. The pilot evaluation of the 6-DMM sensitizing first confirmed a principal possibility of using it for the sensitization of B. sorokiniana and R. solani to triazole- and strobilurin-based fungicides, respectively. Full article

The worldwide spread of antimicrobial resistance highlights the need of new druggable cellular targets. The increasing knowledge of bacterial cell division suggested the potentiality of this pathway as a pool of alternative drug targets, mainly based on the essentiality of these proteins, as well as on the divergence from their eukaryotic counterparts. People suffering from cystic fibrosis are particularly challenged by the lack of antibiotic alternatives. Among the opportunistic pathogens that colonize the lungs of these patients, Burkholderia cenocepacia is a well-known multi-drug resistant bacterium, particularly difficult to treat. Here we describe the organization of its division cell wall (dcw) cluster: we found that 15 genes of the dcw operon can be transcribed as a polycistronic mRNA from mraZ to ftsZ and that its transcription is under the control of a strong promoter regulated by MraZ. B. cenocepacia J2315 FtsZ was also shown to interact with the other components of the divisome machinery, with a few differences respect to other bacteria, such as the direct interaction with FtsQ. Using an in vitro sedimentation assay, we validated the role of SulA as FtsZ inhibitor, and the roles of FtsA and ZipA as tethers of FtsZ polymers. Together our results pave the way for future antimicrobial design based on the divisome as pool of antibiotic cellular targets. Full article

The peptide SET-M33 is a molecule synthesized in tetra-branched form which is being developed as a new antibiotic against Gram-negative bacteria. Its isomeric form with D amino acids instead of the L version (SET-M33D) is also able to kill Gram-positive bacteria because of its higher resistance to bacterial proteases (Falciani et al., PLoS ONE, 2012, 7, e46259). Here we report the strong in vitro activity of SET-M33D (MIC range 0.7–6.0 µM) against multiresistant pathogens of clinical interest, including Gram-positives Staphylococcus aureus, Staphylococcus saprophyticus, and Enterococcus faecalis, and various Gram-negative enterobacteriaceae. SET-M33D antibacterial activity is also confirmed in vivo against a MRSA strain of S. aureus with doses perfectly compatible with clinical use (5 and 2.5 mg/Kg). Moreover, SET-M33D strongly neutralized lipopolysaccharide (LPS) and lipoteichoic acid (LTA), thus exerting a strong anti-inflammatory effect, reducing expression of cytokines, enzymes, and transcription factors (TNF-α, IL6, COX-2, KC, MIP-1, IP10, iNOS, NF-κB) involved in the onset and evolution of the inflammatory process. These results, along with in vitro and in vivo toxicity data and the low frequency of resistance selection reported here, make SET-M33D a strong candidate for the development of a new broad spectrum antibiotic. Full article

China is one of the largest producers of pigs and pork in the world. However, large-scale studies on pig-associated Staphylococcus aureus in relation to healthy pigs, diseased pigs and environment are scarce. The objective of the present study was to characterize and compare S. aureus isolates from healthy pigs, diseased pigs and environment through antimicrobial susceptibility testing, multiple locus sequence typing, spa typing, and antimicrobial resistance gene screening. Results showed all isolates were susceptible to linezolid and vancomycin. However, 66.7% (104/156) isolates were multidrug-resistant by displaying resistance to three or more antibiotics and high rates of resistance to penicillin, tetracycline, clindamycin, and clarithromycin were observed. Of the 20 multilocus sequence types (STs) identified among the isolates, ST9, ST188, and ST7 were most commonly isolated from healthy pigs and environment, while ST1 was most commonly isolated from diseased pigs. In total, 17 spa types were represented among the isolates, while t4792 was most commonly isolated from diseased pigs and t899, t189 were most commonly isolated from healthy pigs and environment. In conclusion, the genotypic and epidemiology characteristics observed among the isolates suggest pigs and pork could be important players in S. aureus dissemination. Full article

Uncontrolled use of drugs both in humans and animals coupled with environmental contamination exacerbate the development and spread of antimicrobial resistance. This paper assessed the drivers of antimicrobial use and resistance in poultry and domestic pig farming and the environment. Questionnaires, in-depth interviews, and focus group discussions (FGDs) were used to collect information regarding demographic characteristics, knowledge, practices, attitudes, and perceptions of the drivers of antimicrobial use and resistance in animal farming and the environment. We found a higher proportion of usage of veterinary antimicrobials for prophylactic purposes (87.6%) in animal farming, than for therapeutic purposes (80.5%). The degree of farming experience was significantly (p < 0.05) related to the knowledge on the source of antimicrobial use, methods used in disease diagnosis, access to veterinary services, stocking of antimicrobials at home, and presence of agriculture activities that involve the use of manure. Uncontrolled disposal of wastes from households, disposal of human and veterinary drugs, and weak implementation of the legal framework was identified as the major contributors to the environment. The high usage of veterinary antimicrobials and the environmental contamination identified requires multisectoral interventions, as well as a review of government strategies, policies, and regulations on antimicrobial use. Full article