In vitro activity of apramycin (EBL-1003) in combination with colistin, meropenem, minocycline or sulbactam against XDR/PDR Acinetobacter baumannii isolates from Greece.

OBJECTIVES: To evaluate the in vitro activity of the combination of apramycin with colistin, meropenem, minocycline or sulbactam, against some well-characterized XDR Acinetobacter baumannii clinical isolates from Greece, to understand how apramycin can be best incorporated into clinical practice and optimize effectiveness. METHODS: In vitro interactions of apramycin (0.5×, 1× and 2× the MIC value) with colistin (2 mg/L), meropenem (30 mg/L), minocycline (3.5 mg/L) or sulbactam (24 mg/L) were tested using time-kill methodology. Twenty-one clinical A. baumannii isolates were chosen, exhibiting apramycin MICs of 4-16 mg/L, which were at or below the apramycin preliminary epidemiological cut-off value of 16 mg/L. These isolates were selected for a range of colistin (4-32 mg/L), meropenem (16-256 mg/L), minocycline (8-32 mg/L) and sulbactam (8-32 mg/L) MICs across the resistant range. Synergy was defined as a ≥2 log10 cfu/mL reduction compared with the most active agent. RESULTS: The combination of apramycin with colistin, meropenem, minocycline or sulbactam was synergistic, at least at one of the concentrations of apramycin (0.5×, 1× or 2× MIC), against 83.3%, 90.5%, 90.9% or 92.3% of the tested isolates, respectively. Apramycin alone was bactericidal at 24 h against 9.5% and 33.3% of the tested isolates at concentrations equal to 1× and 2× MIC, while the combination of apramycin at 2× MIC with colistin, meropenem or sulbactam was bactericidal against all isolates tested (100%). The apramycin 2× MIC/minocycline combination had bactericidal activity against 90.9% of the tested isolates. CONCLUSIONS: Apramycin combinations may have potential as a treatment option for XDR/pandrug-resistant (PDR) A. baumannii infections and warrant validation in the clinical setting, when this new aminoglycoside is available for clinical use.

Fish skin mucosal surface becomes a barrier of antibiotic resistance genes under apramycin exposure.

Antibiotic resistance genes (ARGs) are a kind of emerging environmental contamination, and are commonly found in antibiotic application situations, attracting wide attention. Fish skin mucosal surface (SMS), as the contact interface between fish and water, is the first line of defense against external pollutant invasion. Antibiotics are widely used in aquaculture, and SMS may be exposed to antibiotics. However, what happens to SMS when antibiotics are applied, and whether ARGs are enriched in SMS are not clear. In this study, Zebrafish (Danio rerio) were exposed to antibiotic and antibiotic resistant bacteria in the laboratory to simulate the aquaculture situation, and the effects of SMS on the spread of ARGs were explored. The results showed that SMS maintained the stability of the bacterial abundance and diversity under apramycin (APR) and bacterial exposure effectively. Until 11 days after stopping APR exposure, the abundance of ARGs in SMS (mean value was 3.32 × 10-3 copies/16S rRNA copies) still did not recover to the initial stage before exposure, which means that enriched ARGs in SMS were persistently remained. Moreover, non-specific immunity played an important role in resisting infection of external contamination. Besides, among antioxidant proteins, superoxide dismutase showed the highest activity. Consequently, it showed that SMS became a barrier of antibiotic resistance genes under APR exposure, and ARGs in SMS were difficult to remove once colonized. This study provided a reference for understanding the transmission, enrichment process, and ecological impact of antibiotics and ARGs in aquatic environments.

Apramycin Overcomes the Inherent Lack of Antimicrobial Bactericidal Activity in Mycobacterium abscessus.

Antibiotic therapy of infections caused by the emerging pathogen Mycobacterium abscessus is challenging due to the organism's inherent resistance to clinically available antimicrobials. The low bactericidal potency of currently available treatment regimens is of concern and testifies to the poor therapeutic outcomes for pulmonary M. abscessus infections. Mechanistically, we demonstrate here that the acetyltransferase Eis2 is responsible for the lack of bactericidal activity of amikacin, the standard aminoglycoside used in combination treatment. In contrast, the aminoglycoside apramycin, with a distinct structure, is not modified by any of the pathogen's innate aminoglycoside resistance mechanisms and is not affected by the multidrug resistance regulator WhiB7. As a consequence, apramycin uniquely shows potent bactericidal activity against M. abscessus. This favorable feature of apramycin is reflected in a mouse model of pulmonary M. abscessus infection, which demonstrates superior activity, compared with amikacin. These findings encourage the development of apramycin for the treatment of M. abscessus infections and suggest that M. abscessus eradication in pulmonary disease may be within therapeutic reach.

Clinical Breakpoint of Apramycin to Swine Salmonella and Its Effect on Ileum Flora.

The purpose of this study was to establish the clinical breakpoint (CBP) of apramycin (APR) against Salmonella in swine and evaluate its effect on intestinal microbiota. The CBP was established based on three cutoff values of wild-type cutoff value (COWT), pharmacokinetic-pharmadynamic (PK/PD) cutoff value (COPD) and clinical cutoff value (COCL). The effect of the optimized dose regimen based on ex vivo PK/PD study. The evolution of the ileum flora was determined by the 16rRNA gene sequencing and bioinformatics. This study firstly established the COWT, COPD in ileum, and COCL of APR against swine Salmonella, the value of these cutoffs were 32 µg/mL, 32 µg/mL and 8 µg/mL, respectively. According to the guiding principle of the Clinical Laboratory Standards Institute (CLSI), the final CBP in ileum was 32 µg/mL. Our results revealed the main evolution route in the composition of ileum microbiota of diarrheic piglets treated by APR. The change of the abundances of Bacteroidetes and Euryarchaeota was the most obvious during the evolution process. Methanobrevibacter, Prevotella, S24-7 and Ruminococcaceae were obtained as the highest abundance genus. The abundance of Methanobrevibacter increased significantly when APR treatment carried and decreased in cure and withdrawal period groups. The abundance of Prevotella in the tested groups was significantly lower than that in the healthy group. A decreased of abundance in S24-7 was observed after Salmonella infection and increased slightly after cure. Ruminococcaceae increased significantly after Salmonella infection and decreased significantly after APR treatment. In addition, the genera of Methanobrevibacter and Prevotella were defined as the key node. Valine, leucine and isoleucine biosynthesis, D-Glutamine and D-glutamate metabolism, D-Alanine metabolism, Peptidoglycan and amino acids biosynthesis were the top five Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the ileum microbiota of piglets during the Salmonella infection and APR treatment process. Our study extended the understanding of dynamic shift of gut microbes during diarrheic piglets treated by APR.

Apralogs: Apramycin 5-O-Glycosides and Ethers with Improved Antibacterial Activity and Ribosomal Selectivity and Reduced Susceptibility to the Aminoacyltranserferase (3)-IV Resistance Determinant.

Apramycin is a structurally unique member of the 2-deoxystreptamine class of aminoglycoside antibiotics characterized by a monosubstituted 2-deoxystreptamine ring that carries an unusual bicyclic eight-carbon dialdose moiety. Because of its unusual structure, apramycin is not susceptible to the most prevalent mechanisms of aminoglycoside resistance including the aminoglycoside-modifying enzymes and the ribosomal methyltransferases whose widespread presence severely compromises all aminoglycosides in current clinical practice. These attributes coupled with minimal ototoxocity in animal models combine to make apramycin an excellent starting point for the development of next-generation aminoglycoside antibiotics for the treatment of multidrug-resistant bacterial infections, particularly the ESKAPE pathogens. With this in mind, we describe the design, synthesis, and evaluation of three series of apramycin derivatives, all functionalized at the 5-position, with the goals of increasing the antibacterial potency without sacrificing selectivity between bacterial and eukaryotic ribosomes and of overcoming the rare aminoglycoside acetyltransferase (3)-IV class of aminoglycoside-modifying enzymes that constitutes the only documented mechanism of antimicrobial resistance to apramycin. We show that several apramycin-5-O-ß-d-ribofuranosides, 5-O-ß-d-eryrthofuranosides, and even simple 5-O-aminoalkyl ethers are effective in this respect through the use of cell-free translation assays with wild-type bacterial and humanized bacterial ribosomes and of extensive antibacterial assays with wild-type and resistant Gram negative bacteria carrying either single or multiple resistance determinants. Ex vivo studies with mouse cochlear explants confirm the low levels of ototoxicity predicted on the basis of selectivity at the target level, while the mouse thigh infection model was used to demonstrate the superiority of an apramycin-5-O-glycoside in reducing the bacterial burden in vivo.

Wild-type cutoff for Apramycin against Escherichia coli.

BACKGROUND: Apramycin is used exclusively for the treatment of Escherichia coli (E.coli) infections in swine around the world since the early 1980s. Recently, many research papers have demonstrated that apramycin has significant in vitro activity against multidrug-resistant E.coli isolated in hospitals. Therefore, ensuring the proper use of apramycin in veterinary clinics is of great significance of public health. The objectives of this study were to develop a wild-type cutoff for apramycin against E.coli using a statistical method recommended by Clinical and Laboratory Standards Institute (CLSI) and to investigate the prevalence of resistance genes that confer resistance to apramycin in E. coli. RESULTS: Apramycin susceptibility testing of 1230 E.coli clinical isolates from swine were determinded by broth microdilution testing according to the CLSI document M07-A9. A total number of 310 E.coli strains from different minimum inhibitory concentration (MIC) subsets (0.5-256 µg/mL) were selected for the detection of resistance genes (aac(3)-IV; npmA; apmA) in E. coli by PCR. The percentage of E. coli isolates at each MIC (0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 µg/mL) was 0.08, 0.08, 0.16, 2.93, 31.14, 38.86, 12.85, 2.03, 1.46, and 10.41%. The MIC50 and MIC90 were 16 and 64 µg/mL. All the 310 E.coli isolates were negative for npmA and apmA gene, and only the aac(3)-IV gene was detected in this study. CONCLUSIONS: The wild-type cutoff for apramycin against E.coli was defined as 32 µg/mL. The prevelance of aac(3)-IV gene mainly concentrated in these MIC subsets 'MIC ≥ 64 µg/ mL', which indicates that the wild-type cutoff established in our study is reliable. The wild-type cutoff offers interpretion criteria of apramycin susceptibility testing of E.coli.

Epidemiologic, Phenotypic, and Structural Characterization of Aminoglycoside-Resistance Gene aac(3)-IV.

Aminoglycoside antibiotics are powerful bactericidal therapeutics that are often used in the treatment of critical Gram-negative systemic infections. The emergence and global spread of antibiotic resistance, however, has compromised the clinical utility of aminoglycosides to an extent similar to that found for all other antibiotic-drug classes. Apramycin, a drug candidate currently in clinical development, was suggested as a next-generation aminoglycoside antibiotic with minimal cross-resistance to all other standard-of-care aminoglycosides. Here, we analyzed 591,140 pathogen genomes deposited in the NCBI National Database of Antibiotic Resistant Organisms (NDARO) for annotations of apramycin-resistance genes, and compared them to the genotypic prevalence of carbapenem resistance and 16S-rRNA methyltransferase (RMTase) genes. The 3-N-acetyltransferase gene aac(3)-IV was found to be the only apramycin-resistance gene of clinical relevance, at an average prevalence of 0.7%, which was four-fold lower than that of RMTase genes. In the important subpopulation of carbapenemase-positive isolates, aac(3)-IV was nine-fold less prevalent than RMTase genes. The phenotypic profiling of selected clinical isolates and recombinant strains expressing the aac(3)-IV gene confirmed resistance to not only apramycin, but also gentamicin, tobramycin, and paromomycin. Probing the structure-activity relationship of such substrate promiscuity by site-directed mutagenesis of the aminoglycoside-binding pocket in the acetyltransferase AAC(3)-IV revealed the molecular contacts to His124, Glu185, and Asp187 to be equally critical in binding to apramycin and gentamicin, whereas Asp67 was found to be a discriminating contact. Our findings suggest that aminoglycoside cross-resistance to apramycin in clinical isolates is limited to the substrate promiscuity of a single gene, rendering apramycin best-in-class for the coverage of carbapenem- and aminoglycoside-resistant bacterial infections.

Evaluation of apramycin against spectinomycin-resistant and -susceptible strains of Neisseria gonorrhoeae.

BACKGROUND: The emergence of Neisseria gonorrhoeae resistant to all currently available antimicrobial therapies poses a dire public health threat. New antimicrobial agents with activity against N. gonorrhoeae are urgently needed. Apramycin is an aminocyclitol aminoglycoside with broad-spectrum in vitro activity against MDR Gram-negative pathogens and Staphylococcus aureus. However, its activity against N. gonorrhoeae has not been described. OBJECTIVES: The activity spectrum of apramycin against a collection of MDR N. gonorrhoeae was assessed. Isolates tested included those susceptible and resistant to the structurally distinct aminocyclitol, spectinomycin. RESULTS: The modal MICs for apramycin and spectinomycin were 16 mg/L and 32 mg/L, respectively. The epidemiological cut-off (ECOFF) for apramycin was 64 mg/L. No strains among 77 tested had an MIC above this ECOFF, suggesting very low levels of acquired apramycin resistance. In time-kill analysis, apramycin demonstrated rapid bactericidal activity comparable to that of spectinomycin. CONCLUSIONS: Apramycin has broad-spectrum, rapidly bactericidal activity against N. gonorrhoeae. Future pharmacokinetic and pharmacodynamic studies will be needed to determine whether apramycin and/or apramycin derivatives hold promise as new therapeutics for N. gonorrhoeae infection.

In vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii.

OBJECTIVES: Widespread antimicrobial resistance often limits the availability of therapeutic options to only a few last-resort drugs that are themselves challenged by emerging resistance and adverse side effects. Apramycin, an aminoglycoside antibiotic, has a unique chemical structure that evades almost all resistance mechanisms including the RNA methyltransferases frequently encountered in carbapenemase-producing clinical isolates. This study evaluates the in vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii, and provides a rationale for its superior antibacterial activity in the presence of aminoglycoside resistance determinants. METHODS: A thorough antibacterial assessment of apramycin with 1232 clinical isolates from Europe, Asia, Africa and South America was performed by standard CLSI broth microdilution testing. WGS and susceptibility testing with an engineered panel of aminoglycoside resistance-conferring determinants were used to provide a mechanistic rationale for the breadth of apramycin activity. RESULTS: MIC distributions and MIC90 values demonstrated broad antibacterial activity of apramycin against Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Morganella morganii, Citrobacter freundii, Providencia spp., Proteus mirabilis, Serratia marcescens and A. baumannii. Genotypic analysis revealed the variety of aminoglycoside-modifying enzymes and rRNA methyltransferases that rendered a remarkable proportion of clinical isolates resistant to standard-of-care aminoglycosides, but not to apramycin. Screening a panel of engineered strains each with a single well-defined resistance mechanism further demonstrated a lack of cross-resistance to gentamicin, amikacin, tobramycin and plazomicin. CONCLUSIONS: Its superior breadth of activity renders apramycin a promising drug candidate for the treatment of systemic Gram-negative infections that are resistant to treatment with other aminoglycoside antibiotics.

Efficacy of Apramycin against Multidrug-Resistant Acinetobacter baumannii in the Murine Neutropenic Thigh Model.

Apramycin, an aminocyclitol aminoglycoside, was rapidly bactericidal against Acinetobacter baumannii In a neutropenic murine thigh infection model, treatment-associated A. baumannii CFU reductions of >4 log10 per thigh were observed for all exposures for which area under the curve (AUC)/MIC ratio was >50 and maximum concentration of drug in serum (Cmax)/MIC was ≈10 or higher. Based on these findings, we suggest that apramycin deserves further preclinical exploration as a repurposed therapeutic for multidrug-resistant Gram-negative pathogens, including A. baumannii.

YmdB-mediated down-regulation of sucA inhibits biofilm formation and induces apramycin susceptibility in Escherichia coli.

Antibiotic resistance associated with biofilm formation is a major concern when treating bacterial infections with drugs. The genes and pathways involved in biofilm formation have been extensively studied and are also involved in antibiotic resistance. Recent studies show that overexpression of Escherichia coli (E. coli) YmdB protein alters gene expression profiles and inhibits biofilm formation. Therefore, it is expected that YmdB and its regulated genes play a key role in development of biofilm and antibiotic resistance phenotypes. The present study screened antibiotics to identify those whose susceptibility profiles were regulated by YmdB levels. This protocol identified apramycin. Additional screening for genes negatively regulated by inactivation of RNase III activity via YmdB overexpression revealed that a gene associated with the tricarboxylic acid cycle gene, sucA, was necessary for the YmdB-like phenotype. Taken together, these data suggest that regulation of RNase III activity by trans-acting factors may be the key to identifying genes or pathways connecting biofilm and antibiotic resistance phenotypes. This information could be used to reduce the emergence of biofilm-associated multidrug-resistant bacteria.

Apramycin treatment affects selection and spread of a multidrug-resistant Escherichia coli strain able to colonize the human gut in the intestinal microbiota of pigs.

The effect of apramycin treatment on transfer and selection of an Escherichia coli strain (E. coli 912) in the intestine of pigs was analyzed through an in vivo experiment. The strain was sequenced and assigned to the sequence type ST101 and serotype O11. It carried resistance genes to apramycin/gentamicin, sulphonamide, tetracycline, hygromycin B, ß-lactams and streptomycin [aac(3)-IV, sul2, tet(X), aph(4), bla TEM-1 and strA/B], with all but tet(X) located on the same conjugative plasmid. Nineteen pigs were randomly allocated into two inoculation groups, one treated with apramycin (pen 2) and one non-treated (pen 3), along with a non-inoculated control group (pen 1). Two pigs of pen 2 and 3 were inoculated intragastrically with a rifampicin resistant variant of the strain. Apramycin treatment in pen 2 was initiated immediately after inoculation. Strain colonization was assessed in the feces from all pigs. E. coli 912 was shown to spread to non-inoculated pigs in both groups. The selective effect did not persist beyond 3 days post-treatment, and the strain was not detected from this time point in pen 2. We demonstrated that E. coli 912 was able to spread between pigs in the same pen irrespective of treatment, and apramycin treatment resulted in significantly higher counts compared to the non-treated group. This represents the first demonstration of how antimicrobial treatment affects spread of resistant bacteria in pig production. The use of apramycin may lead to enhanced spread of gentamicin-resistant E. coli. Since gentamicin is a first-choice drug for human bacteremia, this is of concern.

Identification of the molecular attributes required for aminoglycoside activity against Leishmania.

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects millions of people worldwide. Aminoglycosides are mostly known as highly potent, broad-spectrum antibiotics that exert their antibacterial activity by selectively targeting the decoding A site of the bacterial ribosome, leading to aberrant protein synthesis. Recently, some aminoglycosides have been clinically approved and are currently used worldwide for the treatment of leishmaniasis; however the molecular details by which aminoglycosides induce their deleterious effect on Leishmaina is still rather obscure. Based on high conservation of the decoding site among all kingdoms, it is assumed that the putative binding site of these agents in Leishmania is the ribosomal A site. However, although recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics, no such data are presently available regarding their binding site in Leishmania. We present crystal structures of two different aminoglycoside molecules bound to a model of the Leishmania ribosomal A site: Geneticin (G418), a potent aminoglycoside for the treatment of leishmaniasis at a 2.65-Å resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome lacking an antileishmanial activity at 1.4-Å resolution. The structural data, coupled with in vitro inhibition measurements on two strains of Leishmania, provide insight as to the source of the difference in inhibitory activity of different Aminoglycosides. The combined structural and physiological data sets the ground for rational design of new, and more specific, aminoglycoside derivatives as potential therapeutic agents against leishmaniasis.

A type 2 A/C2 plasmid carrying the aacC4 apramycin resistance gene and the erm(42) erythromycin resistance gene recovered from two Salmonella enterica serovars.

OBJECTIVES: To determine the relationships between RepA/C2 plasmids carrying several antibiotic resistance genes found in isolates of Salmonella enterica serovars Ohio and Senftenberg from pigs. METHODS: Illumina HiSeq was used to sequence seven S. enterica isolates. BLAST searches identified relevant A/C2 plasmid contigs and contigs were assembled using PCR. RESULTS: Two serovar Ohio isolates were ST329 and the five Senftenberg isolates were ST210. The A/C2 plasmids recovered from the seven isolates belong to type 2 and contain two resistance islands. Their backbones are closely related, differing by five or fewer SNPs. The sul2-containing resistance island ARI-B is 19.9 kb and also contains the kanamycin and neomycin resistance gene aphA1, the tetracycline resistance gene tetA(D) and an erythromycin resistance gene, erm(42), not previously seen in A/C2 plasmids. A second 30.3 kb resistance island, RI-119, is in a unique location in the A/C2 backbone 8.2 kb downstream of rhs. RI-119 contained genes conferring resistance to apramycin, netilmicin and tobramycin (aacC4), hygromycin (hph), sulphonamides (sul1) and spectinomycin and streptomycin (aadA2). In one of the seven plasmids, this resistance region contained two IS26-mediated deletions. A discrete 5.7 kb segment containing the aacC4 and hph genes and bounded by IS26 on one side and the inverted repeat of Tn5393 on the other was identified. CONCLUSIONS: The presence of almost identical A/C2 plasmids in two serovars indicates a common origin. Type 2 A/C2 plasmids continue to evolve via addition of new resistance regions such as RI-119 and evolution of existing ones.

Apramycin has high in vitro activity against Mycobacterium tuberculosis.

Introduction. Aminoglycoside antibiotics such as amikacin and kanamycin are important components in the treatment of Mycobacterium tuberculosis (Mtb) infection. However, more and more clinical strains are found to be aminoglycoside antibiotic-resistant. Apramycin is another kind of aminoglycoside antibiotic that is commonly used to treat infections in animals.Hypothesis. Apramycin may have in vitro activity against Mtb.Aim. This study aims to evaluate the efficacy of apramycin against Mtb in vitro and determine its epidemiological cut-off (ECOFF) value.Methodology. One hundred Mtb isolates, including 17 pansusceptible and 83 drug-resistant tuberculosis (DR-TB) strains, were analysed for apramycin resistance using the MIC assay.Results. Apramycin exhibited significant inhibitory activity against Mtb clinical isolates, with an MIC50 of 0.5 µg ml-1 and an MIC90 of 1 µg ml-1. We determined the tentative ECOFF value as 1 µg ml-1 for apramycin. The resistant rates of multidrug-resistant tuberculosis (MDR-TB), pre-extensively drug-resistant (pre-XDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains were 12.12 % (4/33), 20.69 % (6/29) and 66.67 % (14/21), respectively. The rrs gene A1401G is associated with apramycin resistance, as well as the cross-resistance between apramycin and other aminoglycosides.Conclusion. Apramycin shows high in vitro activity against the Mtb clinical isolates, especially the MDR-TB clinical isolates. This encouraging discovery calls for more research on the functions of apramycin in vivo and as a possible antibiotic for the treatment of drug-resistant TB.

Apramycin efficacy against carbapenem- and aminoglycoside-resistant Escherichia coli and Klebsiella pneumoniae in murine bloodstream infection models.

BACKGROUND: The aminoglycoside apramycin has been proposed as a drug candidate for the treatment of critical Gram-negative systemic infections. However, the potential of apramycin in the treatment of drug-resistant bloodstream infections (BSIs) has not yet been assessed. METHODS: The resistance gene annotations of 40 888 blood-culture isolates were analysed. In vitro profiling of apramycin comprised cell-free translation assays, broth microdilution, and frequency of resistance determination. The efficacy of apramycin was studied in a mouse peritonitis model for a total of nine Escherichia coli and Klebsiella pneumoniae isolates. RESULTS: Genotypic aminoglycoside resistance was identified in 87.8% of all 6973 carbapenem-resistant Enterobacterales blood-culture isolates, colistin resistance was shown in 46.4% and apramycin in 2.1%. Apramycin activity against methylated ribosomes was > 100-fold higher than that for other aminoglycosides. Frequencies of resistance were < 10-9 at 8 × minimum inhibitory concentration (MIC). Tentative epidemiological cut-offs (TECOFFs) were determined as 8 µg/mL for E. coli and 4 µg/mL for K. pneumoniae. A single dose of 5 to 13 mg/kg resulted in a 1-log colony-forming unit (CFU) reduction in the blood and peritoneum. Two doses of 80 mg/kg resulted in an exposure that resembles the AUC observed for a single 30 mg/kg dose in humans and led to complete eradication of carbapenem- and aminoglycoside-resistant bacteraemia. CONCLUSION: Encouraging coverage and potent in vivo efficacy against a selection of highly drug-resistant Enterobacterales isolates in the mouse peritonitis model warrants the conduct of clinical studies to validate apramycin as a drug candidate for the prophylaxis and treatment of BSI.

Redesign of substrate specificity and identification of the aminoglycoside binding residues of Eis from Mycobacterium tuberculosis.

The upsurge in drug-resistant tuberculosis (TB) is an emerging global problem. The increased expression of the enhanced intracellular survival (Eis) protein is responsible for the clinical resistance to aminoglycoside (AG) antibiotics of Mycobacterium tuberculosis . Eis from M. tuberculosis (Eis_Mtb) and M. smegmatis (Eis_Msm) function as acetyltransferases capable of acetylating multiple amines of many AGs; however, these Eis homologues differ in AG substrate preference and in the number of acetylated amine groups per AG. The AG binding cavity of Eis_Mtb is divided into two narrow channels, whereas Eis_Msm contains one large cavity. Five bulky residues lining one of the AG binding channels of Eis_Mtb, His119, Ile268, Trp289, Gln291, and Glu401, have significantly smaller counterparts in Eis_Msm, Thr119, Gly266, Ala287, Ala289, and Gly401, respectively. To identify the residue(s) responsible for AG binding in Eis_Mtb and for the functional differences from Eis_Msm, we have generated single, double, triple, quadruple, and quintuple mutants of these residues in Eis_Mtb by mutating them into their Eis_Msm counterparts, and we tested their acetylation activity with three structurally diverse AGs: kanamycin A (KAN), paromomyin (PAR), and apramycin (APR). We show that penultimate C-terminal residue Glu401 plays a critical role in the overall activity of Eis_Mtb. We also demonstrate that the identities of residues Ile268, Trp289, and Gln291 (in Eis_Mtb nomenclature) dictate the differences between the acetylation efficiencies of Eis_Mtb and Eis_Msm for KAN and PAR. Finally, we show that the mutation of Trp289 in Eis_Mtb into Ala plays a role in APR acetylation.

In vitro activity of apramycin against 16S-RMTase-producing Gram-negative isolates.

OBJECTIVES: Apramycin is an aminoglycoside (AG) with a unique structure that is little affected by plasmid-mediated mechanisms of AG resistance, including most AG-modifying enzymes and 16S rRNA methyltransferases (16S-RMTases). We evaluate the activity of apramycin against a collection of 16S-RMTase-producing isolates, including Enterobacterales, non-fermenting bacteria, and carbapenemase producers. METHODS: In total, 164 non-duplicate 16S-RMTase-producing isolates, including 84 Enterobacterales, 53 Acinetobacter baumannii and 27 Pseudomonas aeruginosa isolates, were included in the study. Whole-genome sequencing (WGS) was performed on all isolates with Illumina technology. The minimum inhibitory concentration (MIC) of apramycin was determined by broth microdilution with customized Sensititre plates (Thermo Fisher Scientific, Dardilly, France). RESULTS: We found that 95% (156/164) of the 16S-RMTase-producing isolates were susceptible to apramycin, with a MIC50 of 4 mg/L and a MIC90 of 16 mg/L, respectively. Resistance rates were higher in P. aeruginosa (11%) than in A. baumannii (4%) or Enterobacterales (4%) (P < 0.0001 for each comparison). Eight isolates were resistant to apramycin, including one isolate with an MIC >64 mg/L due to the acquisition of the aac(3)-IV gene. The genetic environment of the aac(3)-IV gene was similar to that in the pAH01-4 plasmid of an Escherichia coli isolate from chicken in China. CONCLUSION: Resistance to apramycin remains rare in 16S-RMTase-producing isolates. Apramycin may, therefore, be an interesting alternative treatment for infections caused by 16S-RMTase and carbapenemase producers.

Unusual small plasmids carrying the novel resistance genes dfrK or apmA isolated from methicillin-resistant or -susceptible staphylococci.

OBJECTIVES: The aims of this study were to identify small staphylococcal plasmids that carry either the trimethoprim resistance gene dfrK or the apramycin resistance gene apmA and analyse them for their structure and organization with regard to their potential role as precursors of large multiresistance plasmids that carry these genes. METHODS: Trimethoprim- or apramycin-resistant staphylococci from the strain collections of the two participating institutions were investigated for the presence of plasmid-borne dfrK or apmA genes. The dfrK- or apmA-carrying plasmids were sequenced completely and compared with sequences deposited in the databases. RESULTS: Two small plasmids, the 4957 bp dfrK-carrying plasmid pKKS966 from porcine Staphylococcus hyicus subsp. hyicus and the 4809 bp apmA-carrying plasmid pKKS49 from porcine methicillin-resistant Staphylococcus aureus were identified. Structural analysis revealed that both plasmids had a similar organization, comprising a single resistance gene (dfrK or apmA), a plasmid replication gene (rep) and three partly overlapping genes for mobilization proteins (mobA, mobB and mobC). Comparisons showed 71%-82% amino acid identity between the Rep and Mob proteins of these two plasmids; however, distinctly lesser percentages of identity to Rep and Mob proteins of staphylococci and other bacteria deposited in the databases were detected. CONCLUSIONS: Both plasmids, pKKS966 and pKKS49, appeared not to be typical staphylococcal plasmids. The homology to larger plasmids that harbour the genes apmA and/or dfrK was limited to these resistance genes and their immediate upstream and downstream regions and thus suggested that these small plasmids were not integrated into larger plasmids.

Novel apramycin resistance gene apmA in bovine and porcine methicillin-resistant Staphylococcus aureus ST398 isolates.

A novel apramycin resistance gene, apmA, was detected on the ca.-40-kb resistance plasmid pAFS11 from bovine methicillin-resistant Staphylococcus aureus (MRSA) of sequence type 398 (ST398). The apmA gene coded for a protein of 274 amino acids that was related only distantly to acetyltransferases involved in chloramphenicol or streptogramin A resistance. NsiI deletion of apmA resulted in a 16- to 32-fold decrease in the apramycin MICs. An apmA-specific PCR identified this gene in one additional bovine and four porcine MRSA ST398 isolates.