Resistance to amikacin and other major aminoglycosides is commonly due to enzymatic acetylation by the aminoglycoside 6\'-N-acetyltransferase type I enzyme, of which type Ib [AAC(6\')-Ib] is the most widespread among Gram-negative pathogens. Finding enzymatic inhibitors could be an effective way to overcome resistance and extend the useful life of amikacin. Small molecules possess multiple properties that make them attractive for drug development. Mixture-based combinatorial libraries and positional scanning strategy have led to the identification of a chemical scaffold, pyrrolidine pentamine, that, when substituted with the appropriate functionalities at five locations (R1-R5), inhibits AAC(6\')-Ib-mediated inactivation of amikacin. Structure-activity relationship studies have shown that while truncations to the molecule result in loss of inhibitory activity, modifications of functionalities and stereochemistry have different effects on the inhibitory properties. In this study, we show that alterations at position R1 of the two most active compounds, 2700.001 and 2700.003, reduced inhibition levels, demonstrating the essential nature not only of the presence of an S-phenyl moiety at this location but also the distance to the scaffold. On the other hand, modifications on the R3, R4, and R5 positions had varied effects, demonstrating the potential for optimization. A correlation analysis between molecular docking values (ΔG) and the dose required for two-fold potentiation of the compounds described in this and the previous studies showed a significant correlation between ΔG values and inhibitory activity.

OBJECTIVE: This study was conducted to evaluate the in vitro activity of clinically relevant aminoglycosides and to determine the prevalence of genes encoding aminoglycoside modifying enzymes (AMEs) and 16S ribosomal RNA (rRNA) methyltransferases among aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) clinical isolates. Associated resistances to beta-lactams and their bla genes as well as the genetic relatedness of isolates were also investigated.
METHODS: A total of 105 aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) isolates recovered between March and May 2017 from 100 patients hospitalized in different wards of Charles Nicolle Hospital of Tunis, Tunisia, were studied. Minimal inhibitory concentrations of aminoglycoside compounds were determined by broth microdilution method. Aminoglycosides resistance encoding genes [aph(3´)-Ia, aph(3\') IIa, aph(3´)-VIa, ant(2″)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6\')-Ib, rmtA, rmtB, rmtC, armA, and npmA] and bla genes were investigated by PCR and sequencing. Genetic relatedness was examined by multilocus sequence typing (MLST) for representative isolates.
RESULTS: High rates of aminoglycoside resistance were found: gentamicin (85.7%), tobramycin (87.6%), kanamycin (78.0%), netilmincin (74.3%), and amikcin (18.0%). Most common AME gene was aac(3)-IIa (42%), followed by aac(6\')-Ib (36.2%) and aph(3\')-VIa (32.4%). The majority of isolates were resistant to beta-lactams and blaCTX-M-15 was the most common ESBL. The blaNDM-1 and blaOXA-48 were also produced by 1 and 23 isolates, respectively. Novel sequence types have been reported among our isolates and high-risk clonal lineages have been detected, such as E. coli ST43 (ST131 in Achtman MLST scheme) and K. pneumoniae (ST11/ST13).
CONCLUSIONS: The high prevalence of aminoglycoside resistance rates and the diversity of corresponding genes, with diverse β-lactamase enzymes among genetically heterogeneous clinical isolates present a matter of concern.

The escalating incidence of infective endocarditis (IE) caused by aminoglycoside-resistant Enterococcus is a growing concern for clinicians. This issue is particularly pronounced in elderly patients, who face an elevated risk of renal damage during antibiotic treatment, thereby limiting available pharmacological options. Furthermore, elderly patients often present with multiple comorbidities, leading to heightened mortality rates. In this article, we present a case involving an elderly male patient who sought medical attention on two separate occasions due to inflammation of the lower extremities and lumbosacral pain. Subsequent diagnosis revealed infective endocarditis (IE) caused by high-level gentamicin-resistant Enterococcus faecalis through blood culture and echocardiography. The patient also experienced peripheral and cerebral arterial embolism, secondary spine infection, and subsequent heart failure, highlighting the severity of the clinical situation. Following an initial 10-day course of vancomycin and ceftriaxone therapy, the patient developed renal impairment, necessitating a switch to bactericidal therapy with ampicillin in combination with ceftriaxone. Additionally, aortic valve replacement was performed during this period. Ultimately, the patient achieved clinical remission. This case underscores the critical importance of prompt and accurate diagnosis, appropriate antibiotic selection, and timely surgical intervention in enhancing the prognosis of elderly patients with IE.

UNASSIGNED: Escherichia coli is a significant causative agent of bloodstream infections (BSIs). Aminoglycoside antibiotics play a crucial role in treating severe infections such as sepsis and pneumonia. However, resistance to these antibiotics often occurs due to the production of aminoglycoside-modifying enzymes (AMEs). This study was conducted to assess antimicrobial susceptibility patterns against various aminoglycosides and to determine the prevalence of common AME genes in E. coli strains isolated from BSIs.
UNASSIGNED: Sixty-five E. coli isolates were obtained from blood samples in a referral hospital in Tehran, Iran. The susceptibility patterns of aminoglycosides were determined using disk diffusion method and AMEs genes were investigated using PCR assay.
UNASSIGNED: Resistance to aminoglycosides was observed in 64.6% (42/65) of the isolates. The most frequent resistance rate was found for kanamycin (44.6%) and gentamicin (38.5%), followed by tobramycin (29.2%) and amikacin (4.6%). The most frequent AME gene was aac(3)-IVa, which detected in 49.2% isolates, followed by aac(6)-Ib (40%), aac(3)-IIa (32.3%), and ant(2)-Ia (30.8%), respectively.
UNASSIGNED: Athough the findings of this survey are based on specimens collected from a single hospital, our study shows that the high prevalence of aminoglycoside resistance is primarily attributed to the presence of the aac(3)-Iva, aac(6)-Ib and aac(3)-IIa genes. The low rate of resistance to amikacin makes this antibiotic a good candidate for treatment of BSIs due to E. coli.

Campylobacter jejuni is recognized as a significant foodborne pathogen, and recent studies have indicated a rising trend of aminoglycosides resistance gene aph(2″)-If among C. jejuni isolates from food-producing animals in China. However, systematic information about aph(2″)-If-positive C. jejuni from food-producing animals and other sources worldwide based on whole-genome analysis remains a knowledge gap. In this study, we aimed to analyze the worldwide distribution, genetic environment and phylogenetic tree of aph(2″)-If by utilizing Whole Genome Sequencing (WGS) data obtained, coupled with information in the GenBank database. A total of 160C. jejuni isolates in the GenBank database and 14C. jejuni isolates in our laboratory carrying aph(2″)-If gene were performed for further analysis. WGS analysis revealed the global distribution of aph(2″)-If among C. jejuni from 6 countries. Multilocus Sequence Typing(MLST) results indicated that 70 STs were involved in the dissemination of aph(2″)-If, with ST10086 being the predominant ST. Whole-genome Multilocus Sequence Typing(wg-MLST) analysis according to times, countries, and origins of C. jejuni isolation further demonstrated a close relationship between aph(2″)-If carrying C. jejuni isolates from farm and food. The findings also revealed the existence of 32 distinct types of genetic environments surrounding aph(2″)-If among these isolates. Notably, Type 30, characterized by the arrangement ISsag10-deoD-ant(9)-hp-hp-aph(2″)-If, emerged as the predominant genetic environment. In conclusion, our analysis provides the inaugural perspective on the worldwide distribution of aph(2″)-If. This resistance gene demonstrates horizontal transferability and regional diffusion in a clonal pattern. The close association observed among aph(2″)-If-positive C. jejuni strains isolated from poultry, food, and clinical environments underscores the potential for zoonotic transmission from these isolates.

OBJECTIVE: Staphylococcus aureus is one of the most common pathogens causing bloodstream infection. A rapid characterisation of resistance to methicillin and, occasionally, to aminoglycosides for particular indications, is therefore crucial to quickly adapt the treatment and improve the clinical outcomes of septic patients. Among analytical technologies, targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a promising tool to detect resistance mechanisms in clinical samples.
METHODS: A rapid proteomic method was developed to detect and quantify the most clinically relevant antimicrobial resistance effectors in S. aureus in the context of sepsis: PBP2a, PBP2c, APH(3\')-III, ANT(4\')-I, and AAC(6\')-APH(2\'\'), directly from positive blood cultures and in less than 70 min including a 30-min cefoxitin-induction step. The method was tested on spiked blood culture bottles inoculated with 124 S.aureus, accounting for the known genomic diversity of SCCmec types and the genetic background of the strains.
RESULTS: This method provided 99% agreement for PBP2a (n = 98/99 strains) detection. Agreement was 100% for PBP2c (n = 5/5), APH(3\')-III (n = 16/16), and ANT(4\')-I (n = 20/20), and 94% for AAC(6\')-APH(2\'\') (n = 16/17). Across the entire strain collection, 100% negative agreement was reported for each of the 5 resistance proteins. Additionally, relative quantification of ANT(4\')-I expression allowed to discriminate kanamycin-susceptible and -resistant strains, in all strains harbouring the ant(4\')-Ia gene.
CONCLUSIONS: The LC-MS/MS method presented herein demonstrates its ability to provide a reliable determination of S. aureus resistance mechanisms, directly from positive blood cultures and in a short turnaround time, as required in clinical laboratories.

UNASSIGNED: Aeromonas species have been identified as agents responsible for various diseases in both humans and animals. Multidrug-resistant Aeromonas strains pose a significant public health threat due to their emergence and spread in clinical settings and the environment. The aim of this study was to determine a novel resistance mechanism against aminoglycoside antimicrobials in a clinical isolate.
UNASSIGNED: The function of aac(6\')-Va was verified by gene cloning and antibiotic susceptibility tests. To explore the in vivo activity of the enzyme, recombinant proteins were expressed, and enzyme kinetics were tested. To determine the molecular background and mechanism of aac(6\')-Va, whole-genome sequencing and bioinformatic analysis were performed.
UNASSIGNED: The novel aminoglycoside N-acetyltransferase gene aac(6\')-Va confers resistance to several aminoglycosides. Among the antimicrobials tested, ribostamycin showed the highest increase (128-fold) in the minimum inhibitory concentration (MIC) compared with the control strains. According to the MIC results of the cloned aac(6\')-Va, AAC(6\')-Va also showed the highest catalytic efficiency for ribostamycin [kcat/Km ratio = (3.35 ± 0.17) × 104 M-1 s-1]. Sharing the highest amino acid identity of 54.68% with AAC(6\')-VaIc, the novel aminoglycoside N-acetyltransferase constituted a new branch of the AAC(6\') family due to its different resistance profiles. The gene context of aac(6\')-Va and its close relatives was conserved in the genomes of species of the genus Aeromonas.
UNASSIGNED: The novel resistance gene aac(6\')-Va confers resistance to several aminoglycosides, especially ribostamycin. Our finding of a novel resistance gene in clinical A. hydrophila will help us develop more effective treatments for this pathogen\'s infections.

Aminoglycoside antimicrobials remain valuable therapeutic options, but their effectiveness has been threatened by the production of bacterial 16S ribosomal RNA methyltransferases (16S-RMTases). In this study, we evaluated the genomic epidemiology of 16S-RMTase genes among Gram-negative bacteria circulating in the American continent. A total of 4877 16S-RMTase sequences were identified mainly in Enterobacterales and nonfermenting Gram-negative bacilli isolated from humans, animals, foods, and the environment during 1931-2023. Most of the sequences identified were found in the United States, Brazil, Canada, and Mexico, and the prevalence of 16S-RMTase genes have increased in the last five years (2018-2022). The three species most frequently carrying 16S-RMTase genes were Acinetobacter baummannii, Klebsiella pneumoniae, and Escherichia coli. The armA gene was the most prevalent, but other 16S-RMTase genes (e.g., rmtB, rmtE, and rmtF) could be emerging backstage. More than 90% of 16S-RMTase sequences in the Americas were found in North American countries, and although the 16S-RMTase genes were less prevalent in Central and South American countries, these findings may be underestimations due to limited genomic data. Therefore, whole-genome sequence-based studies focusing on aminoglycoside resistance using a One Health approach in low- and middle-income countries should be encouraged.

The antibiotics overuse for infection treatment was the sparkle in the spreading of multi-drug resistance Acinetobacter baumannii in hospitals. In our study, we evaluated the contribution of the aminoglycoside resistance mechanisms of A. baumannii to the resistance surge in some selected Egyptian hospitals with a checkerboard assay application to retrieve the aminoglycoside activity. The resistance profile analysis of collected 200 A. baumannii isolates revealed a multidrug-resistant pattern with limited susceptibilities to aminoglycosides. Analysis of the prevalence of aminoglycoside-modifying enzyme (AMEs) genes revealed the presence of the six AMEs genes either singly or in combination in selected isolates and aph (3)-VIa gene was the predominant one. At the same time, four efflux pump genes of AdeABC and AdeKJL family showed significant (P < 0.001) up-regulation levels. Moreover, the implementation of combination strategy showed fourteen synergistic activities against two high-level aminoglycoside-resistance (HLAR) A. baumannii isolates. The findings highlighted the alarming levels of aminoglycoside resistance in A. baumannii isolates, which proved that a common enzymatic modification mechanism acts synergistically with decreased antibiotic accumulation in acquiring aminoglycoside resistance. Additionally, the study provides useful information for the promising synergistic combination therapy that reduces the therapeutic dose of aminoglycosides used and subsequently increases their clinical application.

The aminoglycoside antibiotics amikacin, gentamicin, and tobramycin are important therapeutic options for Acinetobacter iinfections. Several genes that confer resistance to one or more of these antibiotics are prevalent in the globally distributed resistant clones of Acinetobacter baumannii, but the aac(6\')-Im (aacA16) gene (amikacin, netilmicin, and tobramycin resistance), first reported in isolates from South Korea, has rarely been reported since. In this study, GC2 isolates (1999 to 2002) from Brisbane, Australia, carrying aac(6\')-Im and belonging to the ST2:ST423:KL6:OCL1 type were identified and sequenced. The aac(6\')-Im gene and surrounds have been incorporated into one end of the IS26-bounded AbGRI2 antibiotic resistance island and are accompanied by a characteristic 70.3-kbp deletion of adjacent chromosome. The compete genome of the 1999 isolate F46 (RBH46) includes only two copies of ISAba1 (in AbGRI1-3 and upstream of ampC) but later isolates, which differ from one another by <10 single nucleotide differences (SND), carry two to seven additional shared copies. Several complete GC2 genomes with aac(6\')-Im in an AbGRI2 island (2004 to 2017; several countries) found in GenBank and two additional Australian A. baumannii isolates (2006) carry different gene sets, KL2, KL9, KL40, or KL52, at the capsule locus. These genomes include ISAba1 copies in a different set of shared locations. The distribution of SND between F46 and AYP-A2, a 2013 ST2:ST208:KL2:OCL1 isolate from Victoria, Australia, revealed that a 640-kbp segment that includes KL2 and the AbGRI1 resistance island replaces the corresponding region in F46. Over 1,000 A. baumannii draft genomes also include aac(6\')-Im, indicating that it is currently globally disseminated and significantly underreported. IMPORTANCE Aminoglycosides are important therapeutic options for treatment of Acinetobacter infections. Here, we show that a little-known aminoglycoside resistance gene, aac(6\')-Im (aacA16), that confers amikacin, netilmicin, and tobramycin resistance has been circulating undetected for many years in a sublineage of A. baumannii global clone 2 (GC2), generally with a second aminoglycoside resistance gene, aacC1, which confers resistance to gentamicin. These two genes are commonly found together in GC2 complete and draft genomes and globally distributed. One isolate appears to be ancestral, as its genome contains few ISAba1 copies, providing insight into the original source of this insertion sequence (IS), which is abundant in most GC2 isolates. Tracking ISAba1 spread can provide a simple means to track the development and ongoing evolution as well as the dissemination of specific lineages and detect the formation of many sublineages. The complete ancestral genome will provide an essential base point for tracking this process.