N-Pyrazinylhydroxybenzamides as biologically active compounds: a hit-expansion study and antimicrobial evaluation.

Background: The development of novel antimicrobial drugs is an essential part of combatting the uprising of antimicrobial resistance. Proper hit-to-lead development is crucially needed. Methods & results: We present a hit-expansion study of N-pyrazinyl- and N-pyridyl-hydroxybenzamides with a comprehensive determination of structure-activity relationships. The antimicrobial screening revealed high selectivity to staphylococci along with antimycobacterial activity with the best value of 6.25 µg/ml against Mycobacterium tuberculosis H37Rv. We proved an inhibition of proteosynthesis and a membrane depolarization of methicillin-resistant Staphylococcus aureus. Conclusion: Our results are a good starting point for further development of new antimicrobial compounds, where the next step would be tuning the potential between relatively nonspecific membrane depolarization effect and specific inhibition of proteosynthesis.

Antimycobacterial pyridine carboxamides: From design to in vivo activity.

Tuberculosis is the number one killer of infectious diseases caused by a single microbe, namely Mycobacterium tuberculosis (Mtb). The success rate of curing this infection is decreasing due to emerging antimicrobial resistance. Therefore, novel treatments are urgently needed. As an attempt to develop new antituberculars effective against both drugs-sensitive and drug-resistant Mtb, we report the synthesis of a novel series inspired by combining fragments from the first-line agents isoniazid and pyrazinamide (series I) and isoniazid with the second-line agent 4-aminosalicylic acid (series II). We identified compound 10c from series II with selective, potent in vitro antimycobacterial activity against both drug-sensitive and drug-resistant Mtb H37Rv strains with no in vitro or in vivo cytotoxicity. In the murine model of tuberculosis, compound 10c caused a statistically significant decrease in colony-forming units (CFU) in spleen. Despite having a 4-aminosalicylic acid fragment in its structure, biochemical studies showed that compound 10c does not directly affect the folate pathway but rather methionine metabolism. In silico simulations indicated the possibility of binding to mycobacterial methionine-tRNA synthetase. Metabolic study in human liver microsomes revealed that compound 10c does not have any known toxic metabolites and has a half-life of 630 min, overcoming the main drawbacks of isoniazid (toxic metabolites) and 4-aminosalicylic acid (short half-life).

Correction: Juhás et al. Improving Antimicrobial Activity and Physico-Chemical Properties by Isosteric Replacement of 2-Aminothiazole with 2-Aminooxazole. Pharmaceuticals 2022, 15, 580.

In the original publication [...].

Design, Synthesis and Antimicrobial Evaluation of New N-(1-Hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)(hetero)aryl-2-carboxamides as Potential Inhibitors of Mycobacterial Leucyl-tRNA Synthetase.

Tuberculosis remains a serious killer among infectious diseases due to its incidence, mortality, and occurrence of resistant mycobacterial strains. The challenge to discover new antimycobacterial agents forced us to prepare a series of N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)(hetero)aryl-2-carboxamides 1-19 via the acylation of 6-aminobenzo[c][1,2]oxaborol-1(3H)-ol with various activated (hetero)arylcarboxylic acids. These novel compounds have been tested in vitro against a panel of clinically important fungi and bacteria, including mycobacteria. Some of the compounds inhibited the growth of mycobacteria in the range of micromolar concentrations and retained this activity also against multidrug-resistant clinical isolates. Half the maximal inhibitory concentrations against the HepG2 cell line indicated an acceptable toxicological profile. No growth inhibition of other bacteria and fungi demonstrated selectivity of the compounds against mycobacteria. The structure-activity relationships have been derived and supported with a molecular docking study, which confirmed a selectivity toward the potential target leucyl-tRNA synthetase without an impact on the human enzyme. The presented compounds can become important materials in antimycobacterial research.

Better Outcome of Off-Label High-Dose Ceftazidime in Hemato-Oncological Patients with Infections Caused by Extensively Drug-Resistant Pseudomonas Aeruginosa.

Background: P. aeruginosa sepsis in immunocompromised patients is a serious complication of cancer treatment, especially in the case of an Extensively Drug Resistant (XDR) pathogen. The aim of the study is to evaluate the efficacy of high-dose ceftazidime in the treatment of XDR P. aeruginosa infection and to compare it with the conventionally treated cohort in hemato-oncological patients. Methods: We identified 27 patients with XDR P. aeruginosa infection during the 2008-2018 period, 16 patients served as a conventionally treated cohort with an antipseudomonal beta-lactam antibiotic in standard dose (cohort A), and 11 patients were treated with high-dose ceftazidime (cohort B). Most of the patients were neutropenic and under active treatment for their cancer in both cohorts. Results: Mortality and related mortality were statistically significantly better for cohort B than cohort A; it was 18.2% and 9.1% for cohort B and 68.8% and 68.8% for cohort A, respectively. More patients in cohort A needed mechanical ventilation and renal replacement therapy, 75% and 50% for cohort A and 27.3% and 9.9% for cohort B, respectively. It corresponded well with the worst sequential organ failure score (SOFA) in cohort A compared to cohort B, 16 versus 7, respectively. Reversible neurotoxicity was seen only in two patients in cohort B. Conclusion: Ceftazidime in high doses is a very potent antibiotic (ATB) for treating XDR P. aeruginosa infections in neutropenic cancer with acceptable toxicity.

Hybridization Approach Toward Novel Antituberculars: Design, Synthesis, and Biological Evaluation of Compounds Combining Pyrazinamide and 4-Aminosalicylic Acid.

Apart from the SARS-CoV-2 virus, tuberculosis remains the leading cause of death from a single infectious agent according to the World Health Organization. As part of our long-term research, we prepared a series of hybrid compounds combining pyrazinamide, a first-line antitubercular agent, and 4-aminosalicylic acid (PAS), a second-line agent. Compound 11 was found to be the most potent, with a broad spectrum of antimycobacterial activity and selectivity toward mycobacterial strains over other pathogens. It also retained its in vitro activity against multiple-drug-resistant mycobacterial strains. Several structural modifications were attempted to improve the in vitro antimycobacterial activity. The δ-lactone form of compound 11 (11') had more potent in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv. Compound 11 was advanced for in vivo studies, where it was proved to be nontoxic in Galleria mellonella and zebrafish models, and it reduced the number of colony-forming units in spleens in the murine model of tuberculosis. Biochemical studies showed that compound 11 targets mycobacterial dihydrofolate reductases (DHFR). An in silico docking study combined with molecular dynamics identified a viable binding mode of compound 11 in mycobacterial DHFR. The lactone 11' opens in human plasma to its parent compound 11 (t1/2 = 21.4 min). Compound 11 was metabolized by human liver fraction by slow hydrolysis of the amidic bond (t1/2 = 187 min) to yield PAS and its starting 6-chloropyrazinoic acid. The long t1/2 of compound 11 overcomes the main drawback of PAS (short t1/2 necessitating frequent administration of high doses of PAS).

Superiority of ceftazidime off-label high-dose regimen in PK/PD target attainment during treatment of extensively drug-resistant Pseudomonas aeruginosa infections in cancer patients.

AIM: The objective of this study was to evaluate off-label high-dose ceftazidime population pharmacokinetics in cancer patients with suspected or proven extensively drug-resistant (XDR) Pseudomonas aeruginosa infections and then to compare the achievement of the pharmacokinetic/pharmacodynamic (PK/PD) target after standard and off-label high-dose regimens using population model-based simulations. A further aim was to clinically observe the occurrence of adverse effects during the off-label high-dose ceftazidime treatment. METHODS: In patients treated with off-label high-dose ceftazidime (3 g every 6 h), blood samples were collected and ceftazidime serum levels measured using LC-MS/MS. A pharmacokinetic population model was developed using a nonlinear mixed-effects modelling approach and Monte Carlo simulations were then used to compare standard and high-dose regimens for PK/PD target attainment. RESULTS: A total of 14 cancer patients with serious infection suspected of XDR P. aeruginosa aetiology were eligible for PK analysis. XDR P. aeruginosa was confirmed in 10 patients as the causative pathogen. Population ceftazidime volume of distribution was 13.23 L, while clearance started at the baseline of 1.48 L/h and increased by 0.0076 L/h with each 1 mL/min/1.73 m2 of eGFR. High-dose regimen showed significantly higher probability of target attainment (i.e., 86% vs. 56% at MIC of 32 mg/L). This was translated into a very low mortality rate of 20%. Only one case of reversible neurological impairment was observed. CONCLUSION: We proved the superiority of the ceftazidime off-label high-dose regimen in PK/PD target attainment with very low occurrence of adverse effects. The off-label high-dose regimen should be used to optimize treatment of XDR P. aeruginosa infections.

High-Pressure Inactivation of Bacillus cereus in Human Breast Milk.

Although Holder pasteurization is the recommended method for processing breast milk, it does affect some of its nutritional and biological properties and is ineffective at inactivating spores. The aim of this study was to find and validate an alternative methodology for processing breast milk to increase its availability for newborn babies and reduce the financial loss associated with discarding milk that has become microbiologically positive. We prepared two series of breast milk samples inoculated with the Bacillus cereus (B. cereus) strain to verify the effectiveness of two high-pressure treatments: (1) 350 MPa/5 min/38 °C in four cycles and (2) cumulative pressure of 350 MPa/20 min/38 °C. We found that the use of pressure in cycles was statistically more effective than cumulative pressure. It reduced the number of spores by three to four orders of magnitude. We verified that the method was reproducible. The routine use of this method could lead to an increased availability of milk for newborn babies, and at the same time, reduce the amount of wasted milk. In addition, high-pressure treatment preserves the nutritional quality of milk.

Adenosine-Mimicking Derivatives of 3-Aminopyrazine-2-Carboxamide: Towards Inhibitors of Prolyl-tRNA Synthetase with Antimycobacterial Activity.

Multidrug-resistant tuberculosis (MDR-TB) poses a significant threat to mankind and as such earned its place on the WHO list of priority pathogens. New antimycobacterials with a mechanism of action different to currently used agents are highly required. This study presents the design, synthesis, and biological evaluation of 3-acylaminopyrazine-2-carboxamides derived from a previously reported inhibitor of human prolyl-tRNA synthetase. Compounds were evaluated in vitro against various strains of mycobacteria, pathogenic bacteria, and fungi of clinical significance. In general, high activity against mycobacteria was noted, while the antibacterial and antifungal activity was minimal. The most active compounds were 4'-substituted 3-(benzamido)pyrazine-2-carboxamides, exerting MIC (Minimum Inhibitory Concentration) from 1.95 to 31.25 µg/mL. Detailed structure-activity relationships were established and rationalized in silico with regard to mycobacterial ProRS as a probable target. The active compounds preserved their activity even against multidrug-resistant strains of Mycobacterium tuberculosis. At the same time, they were non-cytotoxic against HepG2 human hepatocellular carcinoma cells. This project is the first step in the successful repurposing of inhibitors of human ProRS to inhibitors of mycobacterial ProRS with antimycobacterial activity.

Clinical experience with the use of newer antibiotics in internal medicine.

The alarming rise in antibiotic resistance between Gram-positive and Gram-negative bacteria makes maximum use of known and available antibiotics necessary. The aim of this work is to highlight some advantages and disadvantages of antibiotics that have appeared on the market in recent years, and share clinical experience with their use in internal medicine. Flucloxacillin is an antibiotic with a significant antistaphylococcal effect, the most significant indications of the oral form are infections of the skin and soft tissues with the causative agent of Staphylococcus aureus and streptococci. The intravenous variant of flucloxacillin is an noninferior alternative to oxacillin and can be used in severe staphylococcal infections including infective endocarditis. Contributing to the treatment of uncomplicated urinary infections are the oral antibiotics mecilinam and fosfomycin. Their advantages are wide spectrum, good tolerability and possibility to use them in pregnant woman. Other antibiotics expand the treatment options for intravenous treatment of serious infections caused by multidrug-resistant bacteria. Ceftazidime/avibactam is effective for infections caused by Pseudomonas aeruginosa and enterobacteria including producers of broad-spectrum beta-lactamase ESBL, AmpC, KPC and OXA-48. The most important advantage of ceftolozane/ tazobactam is their antipseudomonal effect, is characterized by excellent clinical efficacy even against serious infections caused by Pseudomonas aeruginosa, including some multi-resistant strains.

Improving Antimicrobial Activity and Physico-Chemical Properties by Isosteric Replacement of 2-Aminothiazole with 2-Aminooxazole.

Antimicrobial drug resistance is currently one of the most critical health issues. Pathogens resistant to last-resort antibiotics are increasing, and very few effective antibacterial agents have been introduced in recent years. The promising drug candidates are often discontinued in the primary stages of the drug discovery pipeline due to their unspecific reactivity (PAINS), toxicity, insufficient stability, or low water solubility. In this work, we investigated a series of substituted N-oxazolyl- and N-thiazolylcarboxamides of various pyridinecarboxylic acids. Final compounds were tested against several microbial species. In general, oxazole-containing compounds showed high activity against mycobacteria, especially Mycobacterium tuberculosis (best MICH37Ra = 3.13 µg/mL), including the multidrug-resistant strains. Promising activities against various bacterial and fungal strains were also observed. None of the compounds was significantly cytotoxic against the HepG2 cell line. Experimental measurement of lipophilicity parameter log k'w and water solubility (log S) confirmed significantly (typically two orders in logarithmic scale) increased hydrophilicity/water solubility of oxazole derivatives in comparison with their thiazole isosteres. Mycobacterial ß-ketoacyl-acyl carrier protein synthase III (FabH) was suggested as a probable target by molecular docking and molecular dynamics simulations.

Quantitative Risk Assessment of Bacillus cereus Growth during the Warming of Thawed Pasteurized Human Banked Milk Using a Predictive Mathematical Model.

Bacillus cereus is relatively resistant to pasteurization. We assessed the risk of B. cereus growth during warming and subsequent storage of pasteurized banked milk (PBM) in the warmed state using a predictive mathematical model. Holder pasteurization followed by storage below -18 °C was used. Temperature maps, water activity values, and B. cereus growth in artificially inoculated PBM were obtained during a simulation of manipulation of PBM after its release from a Human Milk Bank. As a real risk level, we chose a B. cereus concentration of 100 CFU/mL; the risk was assessed for three cases: 1. For an immediate post-pasteurization B. cereus concentration below 1 CFU/mL (level of detection); 2. For a B. cereus concentration of 10 CFU/mL, which is allowed in some countries; 3. For a B. cereus concentration of 50 CFU/mL, which is approved for milk formulas. In the first and second cases, no risk was detected after 1 h of storage in the warmed state, while after 2 h of storage, B. cereus concentrations of 102 CFU/mL were occasionally encountered. In the third case, exceeding the B. cereus concentration of 102 CFU/mL could be regularly expected after 2 h of storage. Based on these results, we recommend that post-pasteurization bacteriological analysis be performed as recommended by the European Milk Bank Association (EMBA) and using warmed PBM within 1 h after warming (no exceptions).

A cirrhotic patient in an internal medicine ward.

Liver cirrhosis represents a common condition with substantial mortality. Manifestation and progression of ascites, hepatic encephalopathy or gastrointestinal bleeding are among main reasons for hospital admission. Infections represent another specific area in cirrhotic patients. Timely and correct diagnosis and therapy of these conditions are the mainstay of optimal outcome. Manifestation of complications of liver cirrhosis significantly deteriorates prognosis of the patient. Ascites in portal hypertension develops as a result of sodium and consequently water retention. Therapy comprises of restriction of sodium intake, diuretic therapy with combination of spironolactone and furosemide, alternatively large-volume paracentesis. Hepatic encephalopathy comprises a spectrum of neuropsychiatric abnormalities from subtle changes to overt desorientation and asterixis to hepatic coma. Treatment includes correcting of predisposing conditions, administering of non-absorbable disaccharides or rifaximin. The most common cause of bleeding in a cirrhotic patient is oesophageal bleeding. Therapy is complex including hemodynamic stabilisation, antibiotic prophylaxis, vasoactive and endoscopic treatment. Infections are common causes of decompensation and occurrence of complications of advanced chronic liver disease. Their unfavourable outcome is a result of a complex immune disorder in cirrhotic patients. Specific type of infection in cirrhosis is spontaneous bacterial peritonitis, which has to be always excluded with diagnostic paracentesis. The mainstay of successful therapy of infections is timely and vigorous broad spectrum antibiotic therapy which can significantly improve otherwise unfavourable outcome of these patients.

Design, synthesis and biological evaluation of substituted 3-amino-N-(thiazol-2-yl)pyrazine-2-carboxamides as inhibitors of mycobacterial methionine aminopeptidase 1.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is the number one cause of deaths due to a single infectious agent worldwide. The treatment of TB is lengthy and often complicated by the increasing drug resistance. New compounds with new mechanisms of action are therefore needed. We present the design, synthesis, and biological evaluation of pyrazine-based inhibitors of a prominent antimycobacterial drug target - mycobacterial methionine aminopeptidase 1 (MtMetAP1). The inhibitory activities of the presented compounds were evaluated against the MtMetAP1a isoform, and all derivatives were tested against a broad spectrum of myco(bacteria) and fungi. The cytotoxicity of the compounds was also investigated using Hep G2 cell lines. Overall, high inhibition of the isolated enzyme was observed for 3-substituted N-(thiazol-2-yl)pyrazine-2-carboxamides, particularly when the substituent was represented by 2-substituted benzamide. The extent of inhibition was strongly dependent on the used metal cofactor. The highest inhibition was seen in the presence of Ni2+. Several compounds also showed mediocre in vitro potency against Mtb (both Mtb H37Ra and H37Rv). Despite the structural similarities of bacterial and fungal MetAP1 to mycobacterial MtMetAP1, title compounds did not exert antibacterial nor antifungal activity. The reasons behind the higher activity of 2-substituted benzamido derivatives, as well as the correlation of enzyme inhibition with the in vitro growth inhibition activity is discussed.

Bacillus cereus as a Major Cause of Discarded Pasteurized Human Banked Milk: A Single Human Milk Bank Experience.

A systematic study, performed from 2017-2020 looked at the rate of positive post-pasteurization B. cereus findings, the quantity of B. cereus in pasteurized banked human milk (PBM), and the rate of B. cereus toxicogenic isolates from PBM. During the study period, 6815.71 L (30,943 tested bottles) of PBM were tested, with an average amount per year of 1703.93 L (7736 tested bottles). The PBM discard rate per year due to bacterial contamination varied between 8.7-10.0% and contamination with B. cereus was the most frequent reason. The total number of B. cereus positive tests was 2739 and the proportion of its positivity from all positive tests was between 56.7-66.6%. The prevalence of B. cereus positive tests rose significantly in the summer months. The production of enterotoxin was found in 3 of the 20 tested samples (15.0%). The B. cereus CFU-quantities in the PBM were below 10 CFU/mL in 80% of cases (16 of 20 samples tested). The quantitative data can be used in the risk assessment of cold storage of PBM at temperatures above zero and manipulation of PBM prior to its administration.

Risk Factors for Infections, Antibiotic Therapy, and Its Impact on Cancer Therapy Outcomes for Patients with Solid Tumors.

Infections represent a significant cause of morbidity and mortality in cancer patients. Multiple factors related to the patient, tumor, and cancer therapy can affect the risk of infection in patients with solid tumors. A thorough understanding of such factors can aid in the identification of patients with substantial risk of infection, allowing medical practitioners to tailor therapy and apply prophylactic measures to avoid serious complications. The use of novel treatment modalities, including targeted therapy and immunotherapy, brings diagnostic and therapeutic challenges into the management of infections in cancer patients. A growing body of evidence suggests that antibiotic therapy can modulate both toxicity and antitumor response induced by chemotherapy, radiotherapy, and especially immunotherapy. This article provides a comprehensive review of potential risk factors for infections and therapeutic approaches for the most prevalent infections in patients with solid tumors, and discusses the potential effect of antibiotic therapy on toxicity and efficacy of cancer therapy.

Effect of Restriction of Fluoroquinolone Antibiotics on Clostridioides difficile Infections in the University Hospital Hradec Králové.

Clostridioides difficile is the most common pathogen responsible for hospital-acquired diarrhea. This complication of antibiotic treatment mainly endangers the health of elder patients. Preventing the development of C. difficile infections (CDI) is still a challenge that needs to be addressed. In our study, the results of 872 C. difficile positive stool samples were used to describe the epidemiological situation affected by a change in the prescription of fluoroquinolone antibiotics. In a total, 93 of strains were typed by polymerase chain reaction (PCR) and capillary gel electrophoresis. Between years 2014 and 2018 the decline in the fluoroquinolones consumption was 69.3 defined daily dose (DDD) per 1000 patient-days (from 103.3 to 34.0), in same period CDI incidence declined by 1.3 cases per 10,000 patient-bed days (from 5.6 to 4.3). Results of epidemiologic and statistical analysis shows that decline in fluoroquinolones consumption has significant influence on CDI incidence and prevalence of hypervirulent strains. In the University Hospital Hradec Králové properly managed antibiotic stewardship policy has reduced CDI incidence by 23.2% and lowered rate of hypervirulent ribotypes 001 and 176.

5-Alkylamino-N-phenylpyrazine-2-carboxamides: Design, Preparation, and Antimycobacterial Evaluation.

According to the World Health Organization, tuberculosis is still in the top ten causes of death from a single infectious agent, killing more than 1.7 million people worldwide each year. The rising resistance developed by Mycobacterium tuberculosis against currently used antituberculars is an imperative to develop new compounds with potential antimycobacterial activity. As a part of our continuous research on structural derivatives of the first-line antitubercular pyrazinamide, we have designed, prepared, and assessed the in vitro whole cell growth inhibition activity of forty-two novel 5-alkylamino-N-phenylpyrazine-2-carboxamides with various length of the alkylamino chain (propylamino to octylamino) and various simple substituents on the benzene ring. Final compounds were tested against Mycobacterium tuberculosis H37Ra and four other mycobacterial strains (M. aurum, M. smegmatis, M. kansasii, M. avium) in a modified Microplate Alamar Blue Assay. We identified several candidate molecules with micromolar MIC against M. tuberculosis H37Ra and low in vitro cytotoxicity in HepG2 cell line, for example, N-(4-hydroxyphenyl)-5-(pentylamino)pyrazine-2-carboxamide (3c, MIC = 3.91 µg/mL or 13.02 µM, SI > 38) and 5-(heptylamino)-N-(p-tolyl)pyrazine-2-carboxamide (4e, MIC = 0.78 µg/mL or 2.39 µM, SI > 20). In a complementary screening, we evaluated the in vitro activity against bacterial and fungal strains of clinical importance. We observed no antibacterial activity and sporadic antifungal activity against the Candida genus.

N-Pyrazinoyl Substituted Amino Acids as Potential Antimycobacterial Agents-The Synthesis and Biological Evaluation of Enantiomers.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb), each year causing millions of deaths. In this article, we present the synthesis and biological evaluations of new potential antimycobacterial compounds containing a fragment of the first-line antitubercular drug pyrazinamide (PZA), coupled with methyl or ethyl esters of selected amino acids. The antimicrobial activity was evaluated on a variety of (myco)bacterial strains, including Mtb H37Ra, M. smegmatis, M. aurum, Staphylococcus aureus, Pseudomonas aeruginosa, and fungal strains, including Candida albicans and Aspergillus flavus. Emphasis was placed on the comparison of enantiomer activities. None of the synthesized compounds showed any significant activity against fungal strains, and their antibacterial activities were also low, the best minimum inhibitory concentration (MIC) value was 31.25 µM. However, several compounds presented high activity against Mtb. Overall, higher activity was seen in derivatives containing ʟ-amino acids. Similarly, the activity seems tied to the more lipophilic compounds. The most active derivative contained phenylglycine moiety (PC-ᴅ/ʟ-Pgl-Me, MIC < 1.95 µg/mL). All active compounds possessed low cytotoxicity and good selectivity towards Mtb. To the best of our knowledge, this is the first study comparing the activities of the ᴅ- and ʟ-amino acid derivatives of pyrazinamide as potential antimycobacterial compounds.