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Methicillin-resistant Staphylococcus aureus (MRSA) exemplifies high-level antibiotic resistance in this major human pathogen. Its resistance to chloramphenicol is majorly conferred by enzymatic inactivation via chloramphenicol acetyltransferases (CATs). This modification sterically blocks the antibiotic’s ribosomal binding and thus neutralizes its inhibitory potency. Although CATs have been structurally studied across diverse bacteria species, the structures of S. aureus CATs (saCATs) have remained uncharacterized. To address this gap and elucidate species-specific resistance mechanisms, we determined the first high-resolution crystal structure of saCAT1, the prototypical saCAT enzyme. Structural analysis delineates the active site architecture and reveals the molecular basis for substrate recognition of both chloramphenicol and fusidic acid (FA). Further enzymatic assays demonstrated that the K m value against chloramphenicol is 16.9 µM, and the K i value of the inhibitor FA is 83.7 µM, indicating that the inhibitory capacity of FA is relatively limited. These findings provide an essential structural framework for understanding chloramphenicol resistance in S. aureus and facilitate the rational design of novel antimicrobial strategies to combat multidrug-resistant pathogens.

The biological role of the bacterial chloramphenicol (Chl)–resistance enzyme, chloramphenicol acetyltransferase (CAT), has seen renewed interest due to the resurgent use of Chl against multi-drug-resistant microbes. This looming threat calls for more rationally designed antibiotic derivatives that have improved antimicrobial properties and reduced toxicity in humans. Herein, we utilize native ion mobility spectrometry–mass spectrometry (IMS-MS) to investigate the gas-phase structure and thermodynamic stability of the type I variant of CAT from Escherichia coli ( Ec CAT I ) and several Ec CAT I :ligand-bound complexes. Ec CAT I readily binds multiple Chl without incurring significant changes to its gas-phase structure or stability. A non-hydrolyzable acetyl-CoA derivative (S-ethyl-CoA, S-Et-CoA) was used to kinetically trap Ec CAT I and Chl in a ternary, ligand-bound state ( Ec CAT I :S-Et-CoA:Chl). Using collision-induced unfolding (CIU)-IMS-MS, we find that Chl dissociates from Ec CAT I :S-Et-CoA:Chl complexes at low collision energies, while S-Et-CoA remains bound to Ec CAT I even as protein unfolding occurs. Gas-phase binding constants further suggest that Ec CAT I binds S-Et-CoA more tightly than Chl. Both ligands exhibit negative cooperativity of subsequent ligand binding in their respective binary complexes. While we observe no significant change in structure or stability to Ec CAT I when bound to either or both ligands, we have elucidated novel gas-phase unfolding and dissociation behavior and provided a foundation for further characterization of alternative substrates and/or inhibitors of Ec CAT I . Graphical abstract

A decreased chloramphenicol susceptibility in Haemophilus influenzae is commonly caused by the activity of chloramphenicol acetyltransferases (CATs). However, the involvement of membrane proteins in chloramphenicol susceptibility in H. influenzae remains unclear. In this study, chloramphenicol susceptibility testing, whole-genome sequencing, and analyses of membrane-related genes were performed in 51 H. influenzae isolates. Functional complementation assays and structure-based protein analyses were conducted to assess the effect of proteins with sequence substitutions on the minimum inhibitory concentration (MIC) of chloramphenicol in CAT-negative H. influenzae isolates. Six isolates were resistant to chloramphenicol and positive for type A-2 CATs. Of these isolates, A3256 had a similar level of CAT activity but a higher chloramphenicol MIC relative to the other resistant isolates; it also had 163 specific variations in 58 membrane genes. Regarding the CAT-negative isolates, logistic regression and receiver operator characteristic curve analyses revealed that 48T > G (Asn16Lys), 85 C > T (Leu29Phe), and 88 C > A (Leu30Ile) in HI_0898 (emrA), and 86T > G (Phe29Cys) and 141T > A (Ser47Arg) in HI_1177 (artM) were associated with enhanced chloramphenicol susceptibility, whereas 997G > A (Val333Ile) in HI_1612 (hmrM) was associated with reduced chloramphenicol susceptibility. Furthermore, the chloramphenicol MIC was lower in the CAT-negative isolates with EmrA-Leu29Phe/Leu30Ile or ArtM-Ser47Arg substitution and higher in those with HmrM-Val333Ile substitution, relative to their counterparts. The Val333Ile substitution was associated with enhanced HmrM protein stability and flexibility and increased chloramphenicol MICs in CAT-negative H. influenzae isolates. In conclusion, the substitution in H. influenzae multidrug efflux pump HmrM associated with reduced chloramphenicol susceptibility was characterised.

Acne is a serious disfiguring follicular sebaceous gland disorder that negatively affects patients' quality of life and self-image. Chloramphenicol (CAM) is effective against Propionibacterium acnes and Staphylococcus aureus which cause acne, often used as a hospital preparation for acne treatment. However, because of its toxicity and poor water solubility, its use has been restricted. To overcome these limitations, the study focused on developing CAM-loaded binary ethosomes (CAM-BE) and incorporating them into a hydrogel system for transdermal delivery. CAM-BE were prepared and characterized. Following incorporation of the selected formulation into the hydrogel, the formulation's skin-interaction was evaluated using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and confocal laser scanning microscopy (CLSM). Furthermore, a rat ear acne model was used to evaluate the formulation's in vivo anti-inflammatory efficacy and ex vivo skin permeability. The optimal formulation contained ethanol/propylene glycol ratios of 3:7 (w/w), exhibited particle size was 97.68 ± 4.9 nm, zeta-potential was -23.5 ± 1.3 mV, and encapsulation efficiency was 60.36 ± 2.12%. The BE hydrogel that was created showed persistent drug release. Additionally, it demonstrated an enhanced flow of 4.374 ± 0.12 μg/cm /hour, permeability coefficient was 3.65 ± 0.09 cm/h×10 , and apparent skin deposition was 17.77 ± 1.13 μg/cm . CLSM and ATR-FTIR confirm that loading CAM into a binary ethosomes enables drugs to pass more easily through the stratum corneum. In vivo testing and histopathological analysis demonstrated that the CAM-BE hydrogel significantly inhibited swelling in the rat auricle, compared to both the free CAM hydrogel and adapalene hydrogel. With their strong anti-inflammatory properties and improved skin penetration, binary ethosomes could be a viable new CAM delivery method. The new formulation was therefore seen as quite promising.

Background The environmental gliding bacteria Lysobacter are emerging as a new group of biocontrol agents due to their prolific production of lytic enzymes and potent antibiotic natural products. These bacteria are intrinsically resistant to many antibiotics, but the mechanisms behind the antibiotic resistance have not been investigated. Results Previously, we have used chloramphenicol acetyltransferase gene ( cat ) as a selection marker in genetic manipulation of natural product biosynthetic genes in Lysobacter , because chloramphenicol is one of the two common antibiotics that Lysobacter are susceptible to. Here, we found L. enzymogenes , the most studied species of this genus, could still grow in the presence of a low concentration of chloramphenicol. Three chloramphenicol derivatives ( 1 – 3 ) with an unusual acylation pattern were identified in a cat -containing mutant of L. enzymogenes and in the wild type. The compounds included chloramphenicol 3'-isobutyrate ( 1 ), a new compound chloramphenicol 1'-isobutyrate ( 2 ), and a rare chloramphenicol 3'-isovalerate ( 3 ). Furthermore, a mutation of a global regulator gene ( clp ) or a Gcn5-related N -acetyltransferase (GNAT) gene in L. enzymogenes led to nearly no growth in media containing chloramphenicol, whereas a complementation of clp restored the chloramphenicol acylation as well as antibiotic HSAF production in the clp mutant. Conclusions The results indicated that L. enzymogenes contains a pool of unusual acyl donors for enzymatic modification of chloramphenicol that confers the resistance, which may involve the Clp-GNAT regulatory system. Because Lysobacter are ubiquitous inhabitants of soil and water, the finding may have important implications in understanding microbial competitions and bioactive natural product regulation.

Background The unicellular red alga Cyanidioschyzon merolae exhibits a very simple cellular and genomic architecture. In addition, procedures for genetic modifications, such as gene targeting by homologous recombination and inducible/repressible gene expression, have been developed. However, only two markers for selecting transformants, uracil synthase ( URA ) and chloramphenicol acetyltransferase ( CAT ), are available in this alga. Therefore, manipulation of two or more different chromosomal loci in the same strain in C. merolae is limited. Results This study developed a nuclear targeting and transformant selection system using an antibiotics blasticidin S (BS) and the BS deaminase ( BSD ) selectable marker by homologous recombination in C. merolae . In addition, this study has succeeded in simultaneously modifying two different chromosomal loci by a single-step cotransformation based on the combination of BSD and CAT selectable markers. A C. merolae strain that expresses mitochondrion-targeted mSCARLET (with the BSD marker) and mVENUS (with the CAT marker) from different chromosomal loci was generated with this procedure. Conclusions The newly developed BSD selectable marker enables an additional genetic modification to the already generated C. merolae transformants based on the URA or CAT system. Furthermore, the cotransformation system facilitates multiple genetic modifications . These methods and the simple nature of the C. merolae cellular and genomic architecture will facilitate studies on several phenomena common to photosynthetic eukaryotes.

Chloramphenicol (Cm) and its fluorinated derivative florfenicol (Ff) represent highly potent inhibitors of bacterial protein biosynthesis. As a consequence of the use of Cm in human and veterinary medicine, bacterial pathogens of various species and genera have developed and/or acquired Cm resistance. Ff is solely used in veterinary medicine and has been introduced into clinical use in the mid-1990s. Of the Cm resistance genes known to date, only a small number also mediates resistance to Ff. In this review, we present an overview of the different mechanisms responsible for resistance to Cm and Ff with particular focus on the two different types of chloramphenicol acetyltransferases (CATs), specific exporters and multidrug transporters. Phylogenetic trees of the different CAT proteins and exporter proteins were constructed on the basis of a multisequence alignment. Moreover, information is provided on the mobile genetic elements carrying Cm or Cm/Ff resistance genes to provide a basis for the understanding of the distribution and the spread of Cm resistance – even in the absence of a selective pressure imposed by the use of Cm or Ff.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) infection has increased precipitously over the past several decades, with far-reaching health care and societal costs. MRSA infections in the context of burn wounds lead to invasive disease that could potentially cause mortality. Chloramphenicol is a well-known broad-spectrum bacteriostatic antibiotic that has been used since 1949, but due to its hydrophobicity, poor penetration in skin, fast degradation, and toxicity, its application has been hindered. Furthermore, it has been demonstrated that old antibiotics such as chloramphenicol remained active against a large number of currently prevalent resistant bacterial isolates due to their low-level use in the past. Recently, the novel nanoparticulate drug-delivery system has been used and reported to be exceptionally useful for topical therapeutics, due to its distinctive physical characteristics such as a high surface-to-volume ratio and minuscule size. It helps to achieve better hydrophilicity, bioavailability, and controlled delivery with enhanced therapeutic index, which has resulted in decreased toxicity levels compared to the crude drug. Here, we report a novel chloramphenicol loaded with poly(ε-caprolactone) (PCL)-pluronic composite nanoparticles (CAM-PCL-P NPs), physicochemical characterizations, and its bioactivity evaluation in a MRSA-infected burn-wound animal model. CAM-PCL-P NPs could encapsulate 98.3% of the drug in the nanoparticles and release 81% of the encapsulated drug over 36 days with a time to 50% drug release of 72 hours (51%). Nanoparticle suspensions maintained the initial properties with respect to size and encapsulation efficiency, even after 6 months of storage at 4°C and 25°C, respectively (P>0.05). Significant reduction in the level of toxicity was observed for CAM-PCL-P NPs compared with that of free drug as confirmed from hemolytic activity against human blood erythrocytes and cytotoxicity assay against an MCF-7 breast cancer cell line. In vitro antibacterial activities were performed by zone of inhibition, minimum inhibitory concentrations, minimum bacterial concentration, and time-kill assays, which showed that CAM-PCL-P NPs exhibited significantly enhanced anti-MRSA activity against ten clinical isolates of MRSA strains. The augmented activity of CAM-PCL-P NPs was further tested on a MRSA-infected burn-wound animal model and achieved quicker efficacy in MRSA clearance and improved the survival rate compared with free-chloramphenicol treatment. Thus, we propose CAM-PCL-P NPs as a promising novel antimicrobial candidate that may have a good potential for preclinical applications.

Enhancing the thermostability of thermolabile enzymes extends their practical utility. We previously demonstrated that an error-prone thermophile derived from Geobacillus kaustophilus HTA426 can generate mutant genes encoding enzyme variants that are more thermostable than the parent enzyme. Here, we used this approach, termed as thermoadaptation-directed enzyme evolution, to increase the thermostability of the chloramphenicol acetyltransferase (CAT) of Staphylococcus aureus and successfully generated a CAT variant with an A138T replacement (CATᴬ¹³⁸ᵀ). This variant was heterologously produced, and its enzymatic properties were compared with those of the wild type. We found that CATᴬ¹³⁸ᵀ had substantially higher thermostability than CAT but had comparable activities, showing that the A138T replacement enhanced protein thermostability without affecting the catalytic activity. Because variants CATᴬ¹³⁸S and CATᴬ¹³⁸ⱽ, which were generated via in vitro site-directed mutagenesis, were more thermostable than CAT, the thermostability enhancement resulting from the A138T replacement can be attributed to both the presence of a hydroxyl group and the bulk of the threonine side chain. CATᴬ¹³⁸ᵀ conferred chloramphenicol resistance to G. kaustophilus cells at high temperature more efficiently than CAT. Therefore, the gene encoding CATᴬ¹³⁸ᵀ may be useful as a genetic marker in Geobacillus spp. Notably, CATᴬ¹³⁸ᵀ generation was achieved only by implementing improved procedures (plasmid-based mutations on solid media); previous procedures (chromosome-based mutations in liquid media) were unsuccessful. This result suggests that this improved procedure is crucial for successful thermoadaptation-directed evolution in certain cases and increases the opportunities for generating thermostable enzymes.

The characterization of antibiotic-resistant vibrios isolated from shellfish aquaculture is necessary to elucidate the potential transfer of resistance and to establish effective strategies against vibriosis. With this aim, we analyzed a collection of bacterial isolates obtained from 15 failed hatchery larval cultures that, for the most part, had been treated experimentally with chloramphenicol to prevent vibriosis. Isolates were obtained during a 2-year study from experimental cultures of five different clam species. Among a total of 121 Vibrio isolates studied, 28 were found to be chloramphenicol resistant, suggesting that the shellfish hatchery had been using a sublethal concentration of the antibiotic. Interestingly, chloramphenicol-resistant vibrios showed also resistance to tetracycline and amoxicillin (group A; n=19) or to streptomycin (group B; n=9). Chloramphenicol-resistant vibrios were subjected to a PCR amplification and DNA sequencing of the chloramphenicol acetyltransferase genes (cat), and the same approach was followed to study the tetracycline resistance markers (tet). 16S ribosomal RNA (rRNA) gene sequencing revealed that chloramphenicol-resistant vibrios pertained mostly to the Splendidus clade. Conjugation assays demonstrated that various R-plasmids which harbored the cat II/tet(D) genes and cat III gene in groups A and B respectively, were transferred to E. coli and bivalve pathogenic vibrios. Most interestingly, transconjugants exhibited the antibiotic resistance patterns of the donors, despite having been selected only on the basis of chloramphenicol resistance. This is the first report carried out in a bivalve hatchery elucidating the persistence of resistant vibrios, the mechanisms of antibiotic resistance, and the transfer of different R-plasmids.