The solvent- and surface-dependent adsorption of the lipopeptide antibiotic daptomycin: The general necessity of adsorption tests.

The impact of poor or non-reproducible analyte recoveries due to non-specific drug adsorption on various analytical assays is often underestimated. Even internationally approved guidelines for pharmaceutical analysis such as the EMA guideline on bioanalytical method validation, the ICH guideline M10 on bioanalytical method validation and study sample analysis or the FDA bioanalytical method validation guidance do not adequately encourage more detailed investigations. Furthermore, other areas of research in which the concentration of active pharmaceutical compounds plays a crucial role, for example screening for minimal inhibitory concentrations of bacterial isolates, are potentially affected as well. The aim of this study was to demonstrate the general necessity of drug adsorption tests, using the lipopeptide antibiotic daptomycin as an example. A wide range of typical materials used in processing samples in pharmaceutical and biological analysis, as well as various solvents and biological matrices were included in the experiments. A fully validated LC-MS/MS method was applied for the determination of daptomycin concentrations, which were subsequently used to calculate the recovery. Recovery results (n = 3) ranged from 0.00% to 102.12% with a maximum relative standard deviation of 12.78%. These findings demonstrate that recovery can vary greatly depending on the solvent and the contact material, indicating the need to be optimized and, if applicable, validated. Hence, high reproducibility can only be achieved if all materials (and their manufacturers) used in a method are specified, not just those used in steps considered critical.

Development, validation and application of a selective and sensitive LC-MS/MS method for the quantification of daptomycin in a suspension of Mammaliicoccus sciuri in Mueller-Hinton broth.

Reports of therapy failures related to the use of daptomycin (DAP) are steadily increasing. This is mainly due to emerging DAP resistance for which, however, the underlying mechanism is often unknown. To elucidate the mode of action of novel DAP resistance traits it is indispensable to reliably detect and quantify DAP in complex matrices such as bacterial culture media. In this study, we established a selective and sensitive quantification method for DAP upon growth of a DAP resistant Mammaliicoccus sciuri strain in Mueller-Hinton medium. The method combined methanol-induced protein precipitation followed by high performance liquid chromatography with gradient elution coupled to mass spectrometry (LC-MS/MS) using daptomycin-d5 as internal standard. All validation parameters met the acceptance criteria of the European Medicines Agency (EMA) guideline on bioanalytical method validation. We successfully applied this highly selective and sensitive LC-MS/MS method for the quantification of DAP in in vitro studies addressing DAP resistance mechanisms in Gram-positive bacteria.

Epidemiology and antimicrobial resistance of staphylococci other than Staphylococcus aureus from domestic animals and livestock in Africa: a systematic review.

Introduction: Staphylococci other than Staphylococcus aureus (SOSA) in animals are becoming more pathogenic and antibiotic resistant and can potentially disseminate to humans. However, there is little synthesized information regarding SOSA from animals in Africa. This systematic review provides a comprehensive overview of the epidemiology and antimicrobial resistance of SOSA in companion animals (pets) and livestock in Africa. Method: This systematic review (PROSPERO-CRD42021252303) was conducted according to the PRISMA guidelines, and 75 eligible studies from 13 countries were identified until August 2022. Three electronic databases (Pubmed, Scopus and Web of Science) were employed. Results: The frequently isolated SOSA were S. epidermidis, S. intermedius, S. pseudintermedius, S. xylosus, S. chromogenes, S. hyicus, M. sciuri, S. hominis, and S. haemolyticus. Thirty (40%) studies performed antibiotic susceptibility testing (AST). Penicillin (58%) and tetracycline (28%) resistance were most common across all SOSA with high rates of resistance to aminoglycosides, fluoroquinolones, and macrolides in some species. Resistance to last-resort antibiotics such as linezolid and fusidic acid were also reported. Limited data on strain typing and molecular resistance mechanisms precluded analysis of the clonal diversity of SOSA on the continent. Conclusion: The findings of this review indicate that research on livestock-associated SOSA in Africa is lacking in some regions such as Central and Western Africa, furthermore, research on companion animals and more advanced methods for identification and strain typing of SOSA need to be encouraged. Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier: CRD42021252303.

Farming Practice Influences Antimicrobial Resistance Burden of Non-Aureus Staphylococci in Pig Husbandries.

Non-aureus staphylococci (NAS) are ubiquitous bacteria in livestock-associated environments where they may act as reservoirs of antimicrobial resistance (AMR) genes for pathogens such as Staphylococcus aureus. Here, we tested whether housing conditions in pig farms could influence the overall AMR-NAS burden. Two hundred and forty porcine commensal and environmental NAS isolates from three different farm types (conventional, alternative, and organic) were tested for phenotypic antimicrobial susceptibility and subjected to whole genome sequencing. Genomic data were analysed regarding species identity and AMR gene carriage. Seventeen different NAS species were identified across all farm types. In contrast to conventional farms, no AMR genes were detectable towards methicillin, aminoglycosides, and phenicols in organic farms. Additionally, AMR genes to macrolides and tetracycline were rare among NAS in organic farms, while such genes were common in conventional husbandries. No differences in AMR detection existed between farm types regarding fosfomycin, lincosamides, fusidic acid, and heavy metal resistance gene presence. The combined data show that husbandry conditions influence the occurrence of resistant and multidrug-resistant bacteria in livestock, suggesting that changing husbandry practices may be an appropriate means of limiting the spread of AMR bacteria on farms.

Antimicrobial Resistance Profiles of Coagulase-Negative Staphylococci in Community-Based Healthy Individuals in Germany.

Coagulase-negative staphylococci (CoNS) are common opportunistic pathogens, but also ubiquitous human and animal commensals. Infection-associated CoNS from healthcare environments are typically characterized by pronounced antimicrobial resistance (AMR) including both methicillin- and multidrug-resistant isolates. Less is known about AMR patterns of CoNS colonizing the general population. Here we report on AMR in commensal CoNS recovered from 117 non-hospitalized volunteers in a region of Germany with a high livestock density. Among the 69 individuals colonized with CoNS, 29 had reported contacts to either companion or farm animals. CoNS were selectively cultivated from nasal swabs, followed by species definition by 16S rDNA sequencing and routine antibiotic susceptibility testing. Isolates displaying phenotypic AMR were further tested by PCR for presence of selected AMR genes. A total of 127 CoNS were isolated and Staphylococcus epidermidis (75%) was the most common CoNS species identified. Nine isolates (7%) were methicillin-resistant (MR) and carried the mecA gene, with seven individuals (10%) being colonized with at least one MR-CoNS isolate. While resistance against gentamicin, phenicols and spectinomycin was rare, high resistance rates were found against tetracycline (39%), erythromycin (33%) and fusidic acid (24%). In the majority of isolates, phenotypic resistance could be associated with corresponding AMR gene detection. Multidrug-resistance (MDR) was observed in 23% (29/127) of the isolates, with 33% (23/69) of the individuals being colonized with MDR-CoNS. The combined data suggest that MR- and MDR-CoNS are present in the community, with previous animal contact not significantly influencing the risk of becoming colonized with such isolates.

2-Sulfonylpyrimidines as Privileged Warheads for the Development of S. aureus Sortase A Inhibitors.

Staphylococcus aureus is one of the most frequent causes of nosocomial and community-acquired infections, with emerging multiresistant isolates causing a significant burden to public health systems. We identified 2-sulfonylpyrimidines as a new class of potent inhibitors against S. aureus sortase A acting by covalent modification of the active site cysteine 184. Series of derivatives were synthesized to derive structure-activity relationship (SAR) with the most potent compounds displaying low micromolar KI values. Studies on the inhibition selectivity of homologous cysteine proteases showed that 2-sulfonylpyrimidines reacted efficiently with protonated cysteine residues as found in sortase A, though surprisingly, no reaction occurred with the more nucleophilic cysteine residue from imidazolinium-thiolate dyads of cathepsin-like proteases. By means of enzymatic and chemical kinetics as well as quantum chemical calculations, it could be rationalized that the S N Ar reaction between protonated cysteine residues and 2-sulfonylpyrimidines proceeds in a concerted fashion, and the mechanism involves a ternary transition state with a conjugated base. Molecular docking and enzyme inhibition at variable pH values allowed us to hypothesize that in sortase A this base is represented by the catalytic histidine 120, which could be substantiated by QM model calculation with 4-methylimidazole as histidine analog.

Silence as a way of niche adaptation: mecC-MRSA with variations in the accessory gene regulator (agr) functionality express kaleidoscopic phenotypes.

Functionality of the accessory gene regulator (agr) quorum sensing system is an important factor promoting either acute or chronic infections by the notorious opportunistic human and veterinary pathogen Staphylococcus aureus. Spontaneous alterations of the agr system are known to frequently occur in human healthcare-associated S. aureus lineages. However, data on agr integrity and function are sparse regarding other major clonal lineages. Here we report on the agr system functionality and activity level in mecC-carrying methicillin resistant S. aureus (MRSA) of various animal origins (n = 33) obtained in Europe as well as in closely related human isolates (n = 12). Whole genome analysis assigned all isolates to four clonal complexes (CC) with distinct agr types (CC599 agr I, CC49 agr II, CC130 agr III and CC1943 agr IV). Agr functionality was assessed by a combination of phenotypic assays and proteome analysis. In each CC, isolates with varying agr activity levels were detected, including the presence of completely non-functional variants. Genomic comparison of the agr I-IV encoding regions associated these phenotypic differences with variations in the agrA and agrC genes. The genomic changes were detected independently in divergent lineages, suggesting that agr variation might foster viability and adaptation of emerging MRSA lineages to distinct ecological niches.

Asymmetric Disulfanylbenzamides as Irreversible and Selective Inhibitors of Staphylococcus aureus Sortase A.

Staphylococcus aureus is one of the most frequent causes of nosocomial and community-acquired infections, with drug-resistant strains being responsible for tens of thousands of deaths per year. S. aureus sortase A inhibitors are designed to interfere with virulence determinants. We have identified disulfanylbenzamides as a new class of potent inhibitors against sortase A that act by covalent modification of the active-site cysteine. A broad series of derivatives were synthesized to derive structure-activity relationships (SAR). In vitro and in silico methods allowed the experimentally observed binding affinities and selectivities to be rationalized. The most active compounds were found to have single-digit micromolar Ki values and caused up to a 66 % reduction of S. aureus fibrinogen attachment at an effective inhibitor concentration of 10 µM. This new molecule class exhibited minimal cytotoxicity, low bacterial growth inhibition and impaired sortase-mediated adherence of S. aureus cells.