Mechanisms of Resistance to Aminoglycoside Antibiotics: Overview and Perspectives.

Pseudomonas aeruginosa, a Gram-negative bacterium, is a member of the ESKAPE pathogens and one of the leading causes of healthcare-associated infections worldwide. Aminoglycosides (AGs) are recognized for their efficacy against P. aeruginosa. The most common resistance mechanism against AGs is the acquisition of AG-modifying enzymes (AMEs) by the bacteria, including AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases (ANTs). In this study, we obtained 122 multidrug-resistant P. aeruginosa clinical isolates and evaluated the antibacterial effects of six AGs and two carbapenems alone against all clinical isolates, and in combination against eight selected strains. We further probed for four representatives of the most common AME genes [aac(6')-Ib, aac(3)-IV, ant(2")-Ia, and aph(3')-Ia] by polymerase chain reaction (PCR) and compared the AME patterns of these 122 clinical isolates to their antibiotic resistance profile. Among the diverse antibiotics resistance profile displayed by these clinical isolates, we found correlations between the resistance to various AGs as well as between the resistance to one AG and the resistance to carbapenems. PCR results revealed that the presence of aac(6')-Ib renders these isolates more resistant to a variety of antibiotics. The correlation between resistance to various AGs and carbapenems partially reflects the complex resistance strategies adapted in these pathogens and encourages the development of strategic treatment for each P. aeruginosa infection by considering the genetic information of each isolated bacteria.

Plasmid pMP1-1 in Escherichia coli L-0 encodes aminoglycoside (AG) 3'-phosphotransferase II [APH(3')-II]. This enzyme modifies and confers high-level resistance to kanamycin. Although amikacin is a substrate for APH(3')-II, strain L-0(pMP1-1) is susceptible to amikacin. Plasmid pMP1-2 is a spontaneous mutant of pMP1-1 which determines increased APH(3')-II activity for amikacin, apparently as a result of an increase in the copy number of the plasmid. From amikacin-susceptible, gentamicin-susceptible transformants and transconjugants that bear the APH(3')-II gene on plasmid pMP1-1 or pMP1-2 or cloned into multicopy plasmid pBR322, we selected spontaneous mutants at concentrations of amikacin or gentamicin that were two to four times higher than the MICs of these antibiotics. In each case, whether they were selected by using amikacin or gentamicin, the mutants exhibited modest (two- to eightfold) increases in the MIC of gentamicin and major (64- to 128-fold) increases in the MIC of amikacin. Using these laboratory strains of E. coli, we examined the effects on AG susceptibility of the interaction of AG-modifying enzyme activity and generalized AG uptake. Increasing the level of activity of an AG phosphotransferase in these strains lowered their susceptibility to AGs which were substrates for which the enzyme had low Kms. However, an increase in AG-modifying activity alone did not result in large increases in the MICs for poor substrates of the enzyme. In strains which lacked AG-modifying enzymes, a decrease in the rate of AG uptake increased the MICs modestly for a broad spectrum of AGs. When a strain bore the phosphotransferase, a decrease in generalized AG uptake could raise the MIC further, not only for low-Km substrates, but even for AG substrates for which the enzyme had high Kms. Thus, increased modifying activity, together with a diminished rate of uptake, could produce even higher MICs for poor AG substrates.

Management of multi drug resistant tuberculosis (MDR-TB) patients remains a challenge in the control of tuberculosis worldwide. MDR-TB patients require long term treatment with drugs that cause various adverse effects especially ototoxicity associated with aminoglycoside. Current treatment guidelines still include aminoglycosides use especially in developing countries. However, there is dearth of information regarding ototoxic effects of MDR-TB treatment in Nigeria. This study explored the prevalence of ototoxicity and associated factors among MDR-TB patients during the intensive phase of treatment. A retrospective review of records of 383 MDR-TB patients admitted at two specialist treatment centers in South West Nigeria from 2012-2017 was conducted. Using a structured proforma, socio demographic characteristics and clinical history were extracted. Ototoxicity was assessed as any abnormality reported from a pure tone audiometry. Discrete data were summarized with frequencies, proportions while bivariate analysis was done using chi square test to identify associated factors. Multivariate analysis was performed to identify independent predictors of ototoxic effects. All statistical significance was set at 5%. Out of the 383 records extracted, only 143(37.3%) had audiometry performed. Of these 143 MDR-TB patients, mean age was 36.2 ± 11.3 years and prevalence of any ototoxic effect was 72.7 %. This was higher in older age groups, females and those underweight. (p>0.05) Patients who did not sputum convert at the end of treatment and HIV co infected had higher proportions with ototoxic effects (p < 0.05). However, on multivariate analysis only age remained a significant independent predictor of ototoxic effect (AOR: 8.7; 95% CI: 1.4-52.2). Adverse effects are main deterrents to compliance and completion of long term treatment regimen. High rates of ototoxic effects as seen in this study could affect successful management of MDR-TB patients. Auditory monitoring and rehabilitation should be recommended for MDR-TB patients even after completion of the intensive phase of treatment. Key words: Aminoglycoside ototoxicity, drug resistant tuberculosis, Nigeria.

ACHN-490 is a neoglycoside, or "next-generation" aminoglycoside (AG), that has been identified as a potentially useful agent to combat drug-resistant bacteria emerging in hospitals and health care facilities around the world. A focused medicinal chemistry campaign produced a collection of over 400 sisomicin analogs from which ACHN-490 was selected. We tested ACHN-490 against two panels of Gram-negative and Gram-positive pathogens, many of which harbored AG resistance mechanisms. Unlike legacy AGs, ACHN-490 was active against strains expressing known AG-modifying enzymes, including the three most common such enzymes found in Enterobacteriaceae. ACHN-490 inhibited the growth of AG-resistant Enterobacteriaceae (MIC(90), ≤4 μg/ml), with the exception of Proteus mirabilis and indole-positive Proteae (MIC(90), 8 μg/ml and 16 μg/ml, respectively). ACHN-490 was more active alone in vitro against Pseudomonas aeruginosa and Acinetobacter baumannii isolates with AG-modifying enzymes than against those with altered permeability/efflux. The MIC(90) of ACHN-490 against AG-resistant staphylococci was 2 μg/ml. Due to its promising in vitro and in vivo profiles, ACHN-490 has been advanced into clinical development as a new antibacterial agent.

Aminoglycosides (AGs) are broad-spectrum antibiotics famous for their antibacterial activity against Gram-positive and Gram-negative bacteria, as well as mycobacteria. In the United States, the most prescribed AGs, including amikacin (AMK), gentamicin (GEN), and tobramycin (TOB), are vital components of the treatment for resistant bacterial infections. Arbekacin (ABK), a semisynthetic AG, is widely used for the treatment of resistant Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus in Asia. However, the rapid emergence and development of bacterial resistance are limiting the clinical application of AG antibiotics. Of all bacterial resistance mechanisms against AGs, the acquisition of AG-modifying enzymes (AMEs) by bacteria is the most common. It was previously reported that a variant of a bifunctional AME, the 6'-N-AG acetyltransferase-Ie/2″-O-AG phosphotransferase-Ia [AAC(6')-Ie/APH(2″)-Ia], containing a D80G point mutation and a truncation after amino acid 240 modified ABK and AMK at a new position, the 4‴-amine, therefore displaying a change in regiospecificity. In this study, we aimed to verify the altered regiospecificity of this bifunctional enzyme by mutation and truncation for the potential of derivatizing AGs with chemoenzymatic reactions. With the three variant enzymes in this study that contained either mutation only (D80G), truncation only (1-240), or mutation and truncation (D80G-1-240), we characterized their activity by profiling their substrate promiscuity, determined their kinetics parameters, and performed mass spectrometry to determine how and where ABK and AMK were acetylated by these enzymes. We found that the three mutant enzymes possessed distinct acetylation regiospecificity compared to that of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme and the functional AAC(6')-Ie domain [AAC(6')/APH(2″)-1-194].

SETTING: The ototoxic effects of aminoglycosides (AGs) lead to permanent hearing loss, which is one of the devastating consequences of multidrug-resistant tuberculosis (MDR-TB) treatment. As AG ototoxicity is dose-dependent, the impact of a surrogate measure of AG exposure on AG-induced hearing loss warrants close attention for settings with limited therapeutic drug monitoring.OBJECTIVE: To explore the prognostic impact of cumulative AG dose on AG ototoxicity in patients following initiation of AG-containing treatment for MDR-TB.DESIGN: This prospective cohort study was nested within an ongoing cluster-randomized trial of nurse case management intervention across 10 MDR-TB hospitals in South Africa.RESULTS: The adjusted hazard of AG regimen modification due to ototoxicity in the high-dose group (≥75 mg/kg/week) was 1.33 times higher than in the low-dose group (<75 mg/kg/week, 95%CI 1.09-1.64). The adjusted hazard of developing audiometric hearing loss was 1.34 times higher than in the low-dose group (95%CI 1.01-1.77). Pre-existing hearing loss (adjusted hazard ratio [aHR] 1.71, 95%CI 1.29-2.26) and age (aHR 1.16 per 10 years of age, 95%CI 1.01-1.33) were also associated with an increased risk of hearing loss.CONCLUSION: MDR-TB patients with high AG dose, advanced age and pre-existing hearing loss have a significantly higher risk of AG-induced hearing loss. Those at high risk may be candidates for more frequent monitoring or AG-sparing regimens.

Bacteriophages were discovered just over 100 years ago and have been used to treat bacterial infections in animals since the 1920s. The antimicrobial resistance crisis has led to a new surge of interest in the use of bacteriophage therapy as an alternative or supplement to antimicrobial therapy in humans and other animals. To describe the nature of bacteriophages and provide a critical review and update on the clinical use of bacteriophages in the treatment of challenging bacterial infections, with an emphasis on companion animal veterinary applications. The scientific literature on the subject was critically evaluated. Findings from the most pertinent papers have been presented in summary form and critiqued. Over the last 20 years there has been a considerable increase in the volume and quality of publications dealing with bacteriophage therapy. Some recent papers build on excellent work published in the 1980s and describe promising veterinary applications. Challenges related particularly to the registration and approval of phage remedies will need to be overcome before phage therapy can become a mainstream tool for use in veterinary settings. Considerably more research, particularly controlled clinical trials, needs to be done. Bacteriophage therapy is one of the most promising approaches to tackling the looming antimicrobial resistance crisis, yet substantial regulatory challenges will need to be overcome before it enters widespread use. Phage therapy also may, in the future, improve the management of challenging bacterial infections that are not necessarily multidrug-resistant.

Despite issues with oto/nephrotoxicity and bacterial resistance, aminoglycosides (AGs) remain an effective and widely used class of antibacterial agents. For decades now, efforts toward the development of novel AGs with potential to overcome some of these problems have been major research focuses. 1-N-Acylation, especially γ-amino-β-hydroxybutyrate (AHB) derivatization, has proven to be one of the most successful strategies for improving the overall properties of AGs, including their ability to avoid certain resistance mechanisms. More recently, 6'-N-acylation arose as another possible strategy to improve the properties of these drugs. In this study, we report on the glycinyl, carboxybenzyl, and AHB mono- and diderivatization at the 1-, 6'-, and/or 4‴-amines of the AGs amikacin, kanamycin A, netilmicin, sisomicin, and tobramycin. We also present the antibacterial activities and the reduced reactivity of AG-modifying enzymes (AMEs) toward these new AG derivatives, and identify the AMEs present in the bacterial strains tested.

Counterattacking drug-resistant tuberculosis: molecular strategies and future directions

The development of new aminoglycoside (AG) antibiotics has been required to overcome the resistance mechanism of AG-modifying enzymes (AMEs) of AG-resistant pathogens. The AG acetyltransferase, AAC(6′)-APH(2″), one of the most typical AMEs, exhibiting substrate promiscuity towards a variety of AGs and acyl-CoAs, was employed to enzymatically synthesize new 6′-N-acylated isepamicin (ISP) analogs, 6′-N-acetyl/-propionyl/-malonyl ISPs. They were all active against the ISP-resistant Gram-negative bacteria tested, and the 6′-N-acetyl ISP displayed reduced toxicity compared to ISP in vitro. This study demonstrated the importance of the modification of the 6′-amino group in circumventing AG-resistance and the potential of regioselective enzymatic modification of AG scaffolds for the development of more robust AG antibiotics.

Linezolid for Treating Tuberculosis: A Delicate Balancing Act

Background: Ototoxicity remains one of the major adverse events during multidrug-resistant tuberculosis (MDR-TB) treatment. It is related to the use of aminoglycosides. Bedaquiline offers an opportunity to promote a shorter regimen without aminoglycosides. Methods: This is a retrospective study. We reviewed all the MDR-TB patients with hearing losses for which a 9 month regimen has been applied in 2018 in Kinshasa, DR Congo. Kamamycin and Moxifloxacin had been replaced by Bedaquiline and levofloxacin. Treatment was given ambulatory under dot’s unless the emergencies. Monthly follow-up included clinical and bacteriological features, renal and liver functions, QT Interval. Results: From 184 patients selected for 9 months shorter regimen according to WHO guidelines, 39 had hearing loss (21.2%) and were selected for the study. Mean-age was 35.7 years (Range 18-65), 21 male (54%), 8 (21%) HIV positive, 25 (64%) were under weighted; 26 patients (66.7%) had 50% or less, the 2 lungs fields affected on chest X-ray, and 20 patients (51.2%) with one or more cavities. Sputum smear conversion was respectively 82% and 97.2% at 2 and 4 months. Culture conversion was 86% and 97.2% for the same time. Cultures and Sputum smear remained negative until 9th month for 97.2% patients. Adverse events have been reported by 23 patients (58.9%), but they were severe and very severe only in 2 cases (5.1%). No QT interval over 500 millisecond (ms) noted. Treatment outcomes were: 32 patients cured (82.05%), 3 with treatment completed (7.7%), 3 (7.7%) Died, 1 (2.5%) failure. The follow-up of 6 months after treatment completion did not reveal relapse case. Conclusion: The 9 months shorter regimen with bedaquiline showed a good safety and efficacy with a treatment success of 89.75%. This study shows also that the use of bedaquiline is possible even in low-income environment.

BackgroundPLZ is a next-generation aminoglycoside (AG) that is structurally protected from common AG-modifying enzymes (AMEs) in Enterobacteriaceae and with in vitro activity against multidrug-resistant Enterobacteriaceae, including ESBL-producing, AG-resistant, and carbapenem-resistant isolates. We report microbiological outcomes in the EPIC study, including outcomes for resistant pathogens and by the PLZ MIC.MethodsEPIC was a multinational, randomized, double-blind study in hospitalized patients with cUTI or AP. Patients received IV PLZ (15 mg/kg q24h) or IV MEM (1 g q8h) for 4–7 days, followed by optional oral therapy, for a total of 7–10 days of therapy. The extended mMITT population included patients with ≥1 qualifying baseline pathogen (≥105 CFU/mL urine) who received study drug. Microbiological outcomes were assessed at TOC (day 15–19). Isolate identification and susceptibility testing were conducted by a central laboratory. Whole-genome sequencing was used to identify AME and β-lactamase genes.ResultsOf 609 patients enrolled, 407 (66.8%) were included in the extended mMITT population. The most common uropathogen was Escherichia coli (63.4%) followed by Klebsiella pneumoniae (19.7%). PLZ and MEM MIC50/90 for Enterobacteriaceae were 0.5/2 μg/mL (range: ≤0.06–>128 mg/mL) and 0.015/0.06 mg/mL (range: ≤0.004–128 mg/mL), respectively. ESBL and AG-NS phenotypes were found in 29% and 27% of isolates, respectively. Genotyping detected β-lactamase and AME genes in 32.5% and 36.8% of isolates, respectively, most commonly blaCTX-M-15 (n = 98), blaOXA-1/OXA-30 (n = 82), aac(6′)Ib-cr (n = 79), and aac(3)-IIa (n = 56). Rates of microbiological eradication are shown in Table 1. All Enterobacteriaceae in the PLZ group with a PLZ MIC of 4 µg/mL (6/6) were eradicated at TOC (Table 2). Across 49 patients with concurrent bacteremia, 100% (27/27) and 96% (24/25) of Enterobacteriaceae were cleared from the blood at TOC in the PLZ and MEM groups, respectively.ConclusionPLZ demonstrated comparable or higher microbiological eradication rates compared with MEM for common Gram-negative uropathogens, including resistant pathogens. The results support PLZ as a potential treatment option for cUTI, including AP, caused by Enterobacteriaceae with PLZ MICs of ≤4 mg/mL.DisclosuresT. R. Keepers, Achaogen, Inc.: Employee, Salary. D. S. Cebrik, Achaogen, Inc.: Employee, Salary. D. J. Cloutier, Achaogen, Inc.: Employee and Shareholder, Salary. A. Komirenko, Achaogen, Inc.: Employee and Shareholder, Salary. L. Connolly, Achaogen, Inc.: Consultant, Consulting fee. K. Krause, Achaogen, Inc.: Employee, Salary.

The emergence of antimicrobial resistance (AMR) across bacterial pathogens presents a serious threat to global health. This threat is further exacerbated in tuberculosis (TB), mainly due to a protracted treatment regimen involving a combination of drugs. A diversity of factors contributes to the emergence of drug resistance in TB, which is caused by the pathogen Mycobacterium tuberculosis (Mtb). While the traditional genetic mutation-driven drug resistance mechanisms operate in Mtb, there are also several additional unique features of drug resistance in this pathogen. Research in the past decade has enriched our understanding of such unconventional factors as efflux pumps, bacterial heterogeneity, metabolic states, and host microenvironment. Given that the discovery of new antibiotics is outpaced by the emergence of drug resistance patterns displayed by the pathogen, newer strategies for combating drug resistance are desperately needed. In the context of TB, such approaches include targeting the efflux capability of the pathogen, modulating the host environment to prevent bacterial drug tolerance, and activating the host anti-mycobacterial pathways. In this review, we discuss the traditional mechanisms of drug resistance in Mtb, newer understandings and the shaping of a set of unconventional approaches to target both the emergence and treatment of drug resistance in TB.

Mycobacterium abscessus is innately resistant to a variety of drugs thereby limiting therapeutic options. Bacterial resistance to aminoglycosides (AGs) is conferred mainly by AG-modifying enzymes, which often have overlapping activities. Several putative AG-modifying enzymes are encoded in the genome of M. abscessus . The aim of this study was to investigate the molecular basis underlying AG resistance in M. abscessus . M. abscessus deletion mutants deficient in one of three genes potentially involved in AG resistance, aac(2 ' ) , eis1 and eis2 , were generated by targeted gene inactivation, as were combinatorial double and triple deletion mutants. MICs were determined to study susceptibility to a variety of AG drugs and to capreomycin. Deletion of aac(2 ' ) increased susceptibility of M. abscessus to kanamycin B, tobramycin, dibekacin and gentamicin C. Deletion of eis2 increased susceptibility to capreomycin, hygromycin B, amikacin and kanamycin B. Deletion of eis1 did not affect drug susceptibility. Equally low MICs of apramycin, arbekacin, isepamicin and kanamycin A for WT and mutant strains indicate that these drugs are not inactivated by either AAC(2 ' ) or Eis enzymes. M. abscessus expresses two distinct AG resistance determinants, AAC(2 ' ) and Eis2, which confer clinically relevant drug resistance.