Aminoglycoside-modifying Enzymes Research Articles

Ecology, more than antibiotics consumption, is the major predictor for the global distribution of aminoglycoside-modifying enzymes.

Antibiotic consumption and its abuses have been historically and repeatedly pointed out as the major driver of antibiotic resistance emergence and propagation. However, several examples show that resistance may persist despite substantial reductions in antibiotic use, and that other factors are at stake. Here, we study the temporal, spatial, and ecological distribution patterns of aminoglycoside resistance, by screening more than 160,000 publicly available genomes for 27 clusters of genes encoding aminoglycoside-modifying enzymes (AME genes). We find that AME genes display a very ubiquitous pattern: about 25% of sequenced bacteria carry AME genes. These bacteria were sequenced from all the continents (except Antarctica) and terrestrial biomes, and belong to a wide number of phyla. By focusing on European countries between 1997 and 2018, we show that aminoglycoside consumption has little impact on the prevalence of AME-gene-carrying bacteria, whereas most variation in prevalence is observed among biomes. We further analyze the resemblance of resistome compositions across biomes: soil, wildlife, and human samples appear to be central to understand the exchanges of AME genes between different ecological contexts. Together, these results support the idea that interventional strategies based on reducing antibiotic use should be complemented by a stronger control of exchanges, especially between ecosystems.

Inhibition of Enzymatic Acetylation-Mediated Resistance to Plazomicin by Silver Ions.

Plazomicin is a recent U.S. Food and Drug Administration (FDA)-approved semisynthetic aminoglycoside. Its structure consists of a sisomicin scaffold modified by adding a 2(S)-hydroxy aminobutyryl group at the N1 position and a hydroxyethyl substituent at the 6' position. These substitutions produced a molecule refractory to most aminoglycoside-modifying enzymes. The main enzyme within this group that recognizes plazomicin as substrate is the aminoglycoside 2'-N-acetyltransferase type Ia [AAC(2')-Ia], which reduces the antibiotic's potency. Designing formulations that combine an antimicrobial with an inhibitor of resistance is a recognized strategy to extend the useful life of existing antibiotics. We have recently found that several metal ions inhibit the enzymatic inactivation of numerous aminoglycosides mediated by the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib]. In particular, Ag+, which also enhances the effect of aminoglycosides by other mechanisms, is very effective in interfering with AAC(6')-Ib-mediated resistance to amikacin. Here we report that silver acetate is a potent inhibitor of AAC(2')-Ia-mediated acetylation of plazomicin in vitro, and it reduces resistance levels of Escherichia coli carrying aac(2')-Ia. The resistance reversion assays produced equivalent results when the structural gene was expressed under the control of the natural or the blaTEM-1 promoters. The antibiotic effect of plazomicin in combination with silver was bactericidal, and the mix did not show significant toxicity to human embryonic kidney 293 (HEK293) cells.

Impact of the Recent Clinical and Laboratory Standards Institute Breakpoint Changes on the Antimicrobial Spectrum of Aminoglycosides and the Activity of Plazomicin Against Multidrug-Resistant and Carbapenem-Resistant Enterobacterales From United States Medical Centers.

The Clinical and Laboratory Standards Institute (CLSI) lowered the Enterobacterales-susceptible/-resistant breakpoints for amikacin in 2023 from ≤16/≥64 mg/L to ≤4/≥16 mg/L and the breakpoints for gentamicin and tobramycin from ≤4/≥16 mg/L to ≤2/≥8 mg/L. Because aminoglycosides are frequently used to treat infections caused by multidrug-resistant (MDR) and carbapenem-resistant Enterobacterales (CRE), we evaluated the impact of these changes on the susceptibility rates (%S) of Enterobacterales collected from US medical centers. A total of 9809 Enterobacterales isolates were consecutively collected (1/patient) from 37 US medical centers in 2017-2021 and susceptibility was tested by broth microdilution. Susceptibility rates were calculated using CLSI 2022, CLSI 2023, and US Food and Drug Administration 2022 criteria. Aminoglycoside-nonsusceptible isolates were screened for genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methyltransferases (16RMT). The CLSI breakpoint changes mostly affected amikacin, especially against MDR (94.0%S to 71.0%S), extended-spectrum β-lactamase (ESBL)-producing (96.9%S to 79.7%S), and CRE (75.2%S to 59.0%S) isolates. Plazomicin was active against 96.4% of isolates and retained potent activity against CRE (94.0%S), ESBL-producing (98.9%S), and MDR (94.8%S) isolates. Gentamicin and tobramycin showed limited activity against resistant subsets of Enterobacterales. The AME-encoding genes and 16RMT were observed in 801 (8.2%) and 11 (0.1%) isolates, respectively. Plazomicin was active against 97.3% of the AME producers. The spectrum of activity of amikacin against resistant subsets of Enterobacterales was drastically reduced when interpretative criteria based on pharmacokinetic/pharmacodynamic parameters that are currently used to establish breakpoints for other antimicrobials were applied. Plazomicin was markedly more active than amikacin, gentamicin, or tobramycin against antimicrobial-resistant Enterobacterales.

Klebsiella pneumonia in Sudan: Multidrug Resistance, Polyclonal Dissemination, and Virulence.

The emergence and global expansion of hyper-virulent and multidrug resistant (MDR) Klebsiella pneumoniae is an increasing healthcare threat worldwide. The epidemiology of MDR K. pneumoniae is under-characterized in many parts of the world, particularly Africa. In this study, K. pneumoniae isolates from hospitals in Khartoum, Sudan, have been whole-genome sequenced to investigate their molecular epidemiology, virulence, and resistome profiles. Eighty-six K. pneumoniae were recovered from patients in five hospitals in Khartoum between 2016 and 2020. Antimicrobial susceptibility was performed by disk-diffusion and broth microdilution. All isolates underwent whole genome sequencing using Illumina MiSeq; cgMLST was determined using Ridom SeqSphere+, and 7-loci MLST virulence genes and resistomes were identified. MDR was observed at 80%, with 35 isolates (41%) confirmed carbapenem-resistant. Thirty-seven sequence types were identified, and 14 transmission clusters (TC). Five of these TCs involved more than one hospital. Ybt9 was the most common virulence gene detected, in addition to some isolates harbouring iuc and rmp1. There is a diverse population of K. pneumoniae in Khartoum hospitals, harbouring multiple resistance genes, including genes coding for ESBLs, carbapenemases, and aminoglycoside-modifying enzymes, across multiple ST's. The majority of isolates were singletons and transmissions were rare.

Molecular insights into novel environmental strains of Klebsiella quasipneumoniae harboring different antimicrobial-resistance genes.

The emergence of bacterial pathogens in environmental hosts represents a major risk to public health. This study aimed at characterizing seven novel environmental strains of K. quasipneumoniae using a genomic approach which was misidentified by phenotypic methods in a previous batch of 27 species thought to be K. pneumoniae. Whole-genome sequencing was performed using the Illumina platform, and the generated raw reads were de novo assembled. Comparative genomic, resistome, virulome, mobilome, and phylogeny were then investigated using dierent bioinformatics tools. Six strains were identified as K. quasipneumoniae subsp similipneumoniae and one as K. quasipneumoniae subsp. quasipneumoniae. All isolates were resistant to ampicillin, cephalexin, and amoxicillin-clavulanic acid and harbored the fosA, bla OKP types, oqxB, and oqxA genes. One isolate additionally harbored a gene cassettes consisting of bla SHV-1, bla OXA-1, aac(6')-Ib-cr, catB genes. The aminoglycoside-modifying enzyme gene aph(3")-Ia was bracketed by two insertion elements. Plasmid analyses showed that IncFIBK was the most prevalent plasmid, circulating in six isolates, while one isolate exhibited seven different plasmids. The isolates have virulence genes responsible for capsule formation, lipopolysaccharide, iron uptake aerobactin (iutA), salmochelins (iroE, iroN), enterobactin siderophore, adherence, and biofilm formation (mrkA, mrkB, mrkC, mrkD, mrkF, and mrkH). Our study highlights the ecology and transmission of K. quasipneumoniae (which have the ability to disseminate to other environmental sources including animals) outside the clinical setting and the contribution of water, vegetables, and table surfaces as potential reservoirs of farm-to-fork transmission of disease via local markets in Khartoum, Sudan.

Genotypic Patterns of Multidrug-Resistant Acinetobacter baumannii: A Systematic Review.

Acinetobacter baumannii (A. baumannii) is one of the most common bacteria in nosocomial infections. Inappropriate usage of antibiotics has led to expanding emergence resistance to A. baumannii as a multidrug-resistant (MDR) strain. Empirical antibiotic therapy is necessary to evaluate the resistant gene pattern of MDR A. baumannii. For this purpose, the present study evaluated the resistance genes pattern of MDR A. baumannii collected from hospitalized patients using a genotypic diagnostic technique. To find evidence related to the study objectives, databases were searched such as Google Scholar, Web of Science, Science Direct, PubMed, and Scopus from 2000 to 2022, with specified keywords in the title and text of the articles. Articles were included based on inclusion and exclusion criteria. The mentioned database displayed 284 articles. After screening, 65 eligible articles were included. The results showed that various b-lactamases genes, aminoglycoside-modifying enzymes (AMEs) genes, and pump-expressing genes are resistance gene patterns in MDR A. baumannii isolates. MDR A. baumannii has significantly become resistant to b-lactams, carbapenems, and aminoglycosides.

Genetic relationship, antibiotic resistance pattern and virulence factors of Klebsiella pneunoniae strains isolated from meningitis patients

Backgroud: Meningitis due to Klebsiella pneunoniae is increasingly reported from several Asian countries. Microbiological characteristic of K. pneumoniae strains causing meningitis is not yet explored in Iran. The aim of this study was to demonstrate antibiotic resistance pattern and virulence factors, as well as the genetic relationship of K. pneumoniae strains isolated from meningitis patients. Materials and Mehtods: Eight K. pneumoniae isolates were collected from hospitalized patients at Imam Khomeini hospital, Tehran-Iran from 14 May 2018 to 15 Mar 2020. The antibiotic resistance pattern was determined by disc diffusion method. Antibiotic resistance genes and virulence-associated genes were traced by Polymerase Chain Reaction. Repetitive Extragenic Palindromic Polymerase Chain Reaction was used for evaluation of the genetic relationships among isolates. Findings: Six out of eight isolates were resistant to almost all the 15 antibiotics tested. These six isolates harbored triple antibiotic resistance genes: blaOXA-48, blaSHV, and aac (6’)-Ib. Five of these isolates co-harbored aac (3)-IIa gene. Besides, blaTEM and blaCTXM-1 were detected in five and three isolates respectively. blaNDM-1 was identified in one isolate. All isolates harbored acrAB, ompK36, and tolC resistance genes. None of the isolates were related to K1 or K2 capsular serotypes. The most commonly detected virulence genes were entB (100%), mrkD (100%), ybtS (62.5%) and kfu (25%). rep-PCR fingerprinting discriminated seven isolates into three clusters. Conclusion: Simulations presence of the genes coding for Extended Spectrum Beta Lactamases, Carbapenemases, and Aminoglycoside Modifying Enzymes narrows therapeutic alternatives and imposes a heavy load on the public health system. Antibiotic susceptibility test is recommended however, Carbapenems can still be considered as first-line medications for preliminary empirical treatment of K. pneumoniae meningitis before obtaining susceptibility test results. Low genetic diversity suggests the circulation of certain K. pneumoniae clones in hospital which highlights the establishing of effective infection observation and prevention program.

134. Characterization of circulating clinical multi-drug resistant and methicillin resistant Staphylococcus aureus isolates in Peru

Abstract Background Multi-drug resistant and methicillin resistant Staphylococcus aureus (MDR-SA and MRSA, respectively) constitute a significant threat to hospitalized patients. Surveillance for antibiotic resistance is needed in settings where over-the-counter antibiotic use is widespread to identify increasing antibiotic resistance that limits treatment options of nosocomial infections. Methods We performed phenotypic and genotypic characterizations of MDR-SA prospectively obtained from hospitals in Lima, Peru between 2015 - 2018. We collected 3103 bacterial isolates from clinical specimens. Bacterial samples were identified by conventional microbiology and BD Phoenix, and antimicrobial susceptibility testing (AST) was performed by disc diffusion, E-test and BD Phoenix. Additionally, multiplex PCR detected mecA, femA, and lukS in MDR-SA isolates. We selected 80 S. aureus isolates that were resistant to ≥ 3 antimicrobial classes and/or resistant to oxacillin or cefoxitin for genomic characterization using an Illumina NextSeq 500 sequencer. Results Testing identified 232/3103 isolates as S. aureus, and 80/232 (34%) isolates were MDR-SA, resistant to up to nine different antimicrobial classes. We observed high proportions of resistance to aminopenicillins 80/80 (100%), macrolides 71/80 (89%), lincomycins 69/80 (86%), quinolones 66/80 (83%), aminoglycosides 38/46 (82%). Notably, 75/80 (94%) isolates were MRSA. Interestingly, AST revealed that 81% had inducible resistance to clindamycin. In contrast, all isolates were susceptible to vancomycin, linezolid, and daptomycin. Multiplex PCR revealed that 5% of isolates carried the lukS gene and the mecA gene. 76 % of the isolates carried a bifunctional aminoglycoside modifying enzyme and a aminoglycoside O-nucleotidyltransferase enzymes that confer resistance to aminoglycosides. We detected the ermA gene in 79% of the isolates. In silico multilocus sequence typing (MLST) assigned the sequence type 5 to 91.2% MRSA isolates with multiple high related clusters observed, indicating the possibility of nosocomial dissemination and possible outbreak events. Conclusion Antibiotic pressure on the resistance patterns of S. aureus is evident in the high levels of resistance seen in these clinical isolates. Disclosures All Authors: No reported disclosures.

Molecular Epidemiology, Antimicrobial Susceptibility, and Clinical Features of Methicillin-Resistant Staphylococcus aureus Bloodstream Infections over 30 Years in Barcelona, Spain (1990-2019).

Methicillin-resistant Staphylococcus aureus bloodstream infections (MRSA-BSI) are a significant cause of mortality. We analysed the evolution of the molecular and clinical epidemiology of MRSA-BSI (n = 784) in adult patients (Barcelona, 1990−2019). Isolates were tested for antimicrobial susceptibility and genotyped (PFGE), and a selection was sequenced (WGS) to characterise the pangenome and mechanisms underlying antimicrobial resistance. Increases in patient age (60 to 71 years), comorbidities (Charlson’s index > 2, 10% to 94%), community-onset healthcare-associated acquisition (9% to 60%), and 30-day mortality (28% to 36%) were observed during the 1990−1995 and 2014−2019 periods. The proportion of catheter-related BSIs fell from 57% to 20%. Current MRSA-BSIs are caused by CC5-IV and an upward trend of CC8-IV and CC22-IV clones. CC5 and CC8 had the lowest core genome proportions. Antimicrobial resistance rates fell, and only ciprofloxacin, tobramycin, and erythromycin remained high (>50%) due to GyrA/GrlA changes, the presence of aminoglycoside-modifying enzymes (AAC(6′)-Ie-APH(2″)-Ia and ANT(4′)-Ia), and mph(C)/msr(A) or erm (C) genes. Two CC22-IV strains showed daptomycin resistance (MprF substitutions). MRSA-BSI has become healthcare-associated, affecting elderly patients with comorbidities and causing high mortality rates. Clonal replacement with CC5-IV and CC8-IV clones resulted in lower antimicrobial resistance rates. The increased frequency of the successful CC22-IV, associated with daptomycin resistance, should be monitored.

Prevalence of Genes Encoding Resistance to Aminoglycosides and Virulence Factors Among Intestinal Vancomycin-Resistant Enterococci

Background: Vancomycin-resistant enterococci (VRE) are recognized as nosocomial pathogens with increased importance in recent years. These bacteria are frequently isolated from patients admitted to intensive care units (ICUs). Enterococcal pathogenicity is enhanced by different antibiotic resistance and virulence determinants. Objectives: The present study aimed to assess the prevalence of genes encoding resistance to antibiotics and virulence factors in intestinal VRE isolates from ICU patients. Methods: In this study, 23 VREs were investigated. Minimum inhibitory concentrations (MICs) to nine antimicrobial agents were examined using E-test. Genes encoding vancomycin resistance (vanABCDMN), aminoglycoside-modifying enzymes (aac(6')-Ie-aph(2")-Ia, aph(2")-Ib, aph(2")-Ic, aph(2")-Id, aph(3')-IIIa, ant(3')-Ia, ant(4')-Ia, ant(6')-Ia), together with genes for various virulence factor (ace/acm, asa1, cylA, efaA, esp, gelE and hyl), were detected using multiplex PCR. Results: The species distribution of the tested VRE was as follows: Nine Enterococcus casseliflavus, seven E. gallinarum, and seven E. faecium. The vanA gene was found in all E. faecium, in six of which the classical VanA phenotype was observed. The vancomycin (vanC) phenotype was associated with the presence of vanC1 gene in E. gallinarum and the vanC2 gene in E. casseliflavus isolates. The aac(6')-Ie-aph(2")-Ia gene was encoding high-level gentamicin resistance (HLGR) in the studied VRE. All E. faecium were positive for acm and esp, while acm in combination with esp or hyl was detected in 2 vanC enterococci. Conclusions: According to the findings, there was a correlation between the phenotype and the genotype of glycopeptide resistance in the tested VRE. HLGR was more prevalent in E. faecium because of the presence of aac(6')-Ie-aph(2")-Ia. The higher prevalence of virulence determinants was confirmed in vanA isolates compared to the studied vanC-carrying enterococci.

Aminoglycoside resistance in domestic sewage and clinical Escherichia coli isolates

Introduction: Escherichia coli, a Gram-negative bacillus, is found in diverse environmentsand causes several human diseases, such as pneumonia and urinary tractinfections. Aminoglycosides are antimicrobials that present high activity againstGram-negative species, including multidrug-resistant pathogens. However, theindiscriminate use of these compounds has selected resistant microorganisms, mainlydue to the production of aminoglycoside-modifying enzymes (AME). Material andmethods: The minimal inhibitory concentration of the aminoglycosides amikacin,gentamicin, and neomycin against clinical (CI, n = 52, only urinary) and domesticsewage (DS, n = 33) E. coli isolates was determined by the microdilution method,according to the European Committee on Antimicrobial Susceptibility Testing. Thepresence of AMEs among E. coli isolates was determined based on the susceptibilityprofile to amikacin, gentamicin, kanamycin, and tobramycin, according to Manciniet al. (2019). Results: Overall, 33.3% of the DS isolates and 100% of the CIisolates presented mechanisms of resistance to amikacin, gentamicin, or neomycin.The extended-spectrum beta-lactamase enzymes-producing isolates (23/27, 85%)showed mechanisms of resistance to gentamicin and/or neomycin and resistanceto amikacin was simultaneously observed only in CI isolates. All DS isolates were considered wild-type-no AME, while APH (3’) (14/52) and AAC (3’) (10/52)enzymes were detected among CI isolates, one of which produces APH (3’) andAAC (6’)-I simultaneously. Conclusion: Resistance to aminoglycosides is presentamong E. coli isolates in Brazil, but to a lesser extent in environmental isolates.Besides, AMEs are frequent in CI isolates, and surveillance for antimicrobial resistanceshould be implemented to monitor aminoglycoside-resistant E. coli infections.

Design, Synthesis, and Bioassay of 2'-Modified Kanamycin A.

Chemical modification of old drugs is an important way to obtain new ones, and it has been widely used in developing new aminoglycoside antibiotics. However, many of the previous modifying strategies seem arbitrary for their lack of support from structural biological detail. In this paper, based on the structural information of aminoglycoside and its drug target, we firstly analyzed the reason that some 2'-N-acetylated products of aminoglycosides caused by aminoglycoside-modifying enzyme AAC(2') can partially retain activity, and then we designed, synthesized, and evaluated a series of 2'-modified kanamycin A derivatives. Bioassay results showed our modifying strategy was feasible. Our study provided valuable structure-activity relationship information, which would help researchers to develop new aminoglycoside antibiotics more effectively.

Artemisinin derivative DHA27 enhances the antibacterial effect of aminoglycosides against Pseudomonas aeruginosa by inhibiting mRNA expression of aminoglycoside-modifying enzymes.

Bacterial resistance is becoming increasingly serious, the present study aimed to investigate the mechanism of antibacterial sensitization effect of DHA27 combined with tobramycin in tobramycin-resistant Pseudomonas aeruginosa (PA). We found that DHA27 combined with aminoglycosides had an antibacterial sensitization effect on PA. Tobramycin, owing to its lower toxic and side effects, was selected to further study the molecular mechanism of drug combination. A sublethal-dose bacterial challenge/sepsis mouse model was established to study the protective effect of DHA27 plus tobramycin. Scanning electron microscopy was used to investigate whether DHA27 exerts the antibacterial sensitization effect by directly affecting bacterial morphology. The effect of DHA27 on daunorubicin accumulation in bacteria was studied, and quantitative reverse transcription PCR was used to study the effect of DHA27 plus tobramycin on 16S rRNA methyltransferase and aminoglycoside-modifying enzyme mRNA expression. Twenty clinical isolates of PA were found to be tobramycin resistant; DHA27 plus tobramycin had a significant antibacterial sensitization effect on many of these resistant strains. DHA27 plus tobramycin reduced the bacterial load in the spleen and lungs of sepsis model mice and levels of proinflammatory cytokines interleukin-1β (IL-1β) and interferon-γ (IFN-γ). DHA27 plus tobramycin significantly inhibited the mRNA expression of aminoglycoside-modifying enzymes in bacteria. DHA27 combined with AGs had an antibacterial sensitization effect on PA; the molecular mechanism underlying this effect is closely related to the inhibition of the mRNA expression of aminoglycoside-modifying enzymes, especially aac(3)-II.

Acquisition of Streptomycin Resistance by Oxidative Stress Induced by Hydrogen Peroxide in Radiation-Resistant Bacterium Deinococcus geothermalis.

Streptomycin is used primarily to treat bacterial infections, including brucellosis, plague, and tuberculosis. Streptomycin resistance easily develops in numerous bacteria through the inhibition of antibiotic transfer, the production of aminoglycoside-modifying enzymes, or mutations in ribosomal components with clinical doses of streptomycin treatment. (1) Background: A transposable insertion sequence is one of the mutation agents in bacterial genomes under oxidative stress. (2) Methods: In the radiation-resistant bacterium Deinococcus geothermalis subjected to chronic oxidative stress induced by 20 mM hydrogen peroxide, active transposition of an insertion sequence element and several point mutations in three streptomycin resistance (SmR)-related genes (rsmG, rpsL, and mthA) were identified. (3) Results: ISDge6 of the IS5 family integrated into the rsmG gene (dgeo_2335), called SrsmG, encodes a ribosomal guanosine methyltransferase resulting in streptomycin resistance. In the case of dgeo_2840-disrupted mutant strains (S1 and S2), growth inhibition under antibiotic-free conditions was recovered with increased growth yields in the presence of 50 µg/mL streptomycin due to a streptomycin-dependent (SmD) mutation. These mutants have a predicted proline-to-leucine substitution at the 91st residue of ribosomal protein S12 in the decoding center. (4) Conclusions: Our findings show that the active transposition of a unique IS element under oxidative stress conditions conferred antibiotic resistance through the disruption of rsmG. Furthermore, chronic oxidative stress induced by hydrogen peroxide also induced streptomycin resistance caused by point and frameshift mutations of streptomycin-interacting residues such as K43, K88, and P91 in RpsL and four genes for streptomycin resistance.

Evaluation of aminoglycoside modifying enzymes, SCCmec, coagulase gene and PCR-RFLP coagulase gene typing of Staphylococcus aureus isolates from hospitals in Shiraz, southwest of Iran

Staphylococcus aureus is an important human pathogen that causes various infections. Aminoglycosides are broad-spectrum antibiotics used to treat methicillinresistant S. aureus (MRSA) infections. Typing of S. aureus isolates by coagulase gene typing and PCR-RFLP coa gene is a fast and suitable method for epidemiological studies. Aim of the present study was to evaluate the resistance to aminoglycosides, staphylococcal chromosomal cassette mec (SCCmec) types, coagulation typing and PCR-RFLP coa gene in clinical isolates of S. aureus. 192 S. aureus isolates were collected from Namazi and Shahid Faghihi hospitals. Antibiotic resistance was measured by disk diffusion method and MIC was determined for gentamicin. The presence of genes encoding aminoglycoside modifying enzymes (AME) and mecA gene were assessed by PCR. Also the coagulase typing, PCR-RFLP coa gene, and SCCmec typing were performed. Out of 192 isolated S. aureus isolates, 83 (43.2%) MRSA isolates were identified. In this study, a high resistance to streptomycin and gentamicin (98.7%) were observed. Among the AME genes, the aac (6′)-Ie-aph (2″) gene was the most common. Based on the SCCmec typing, it was determined that the prevalence of SCCmec type III (45.8%) was highest. From the amplification of the coa gene, 5 different types were obtained. Also, in digestion of coa gene products by HaeIII enzyme, 10 different RFLP patterns were observed. According to this study, aminoglycoside resistance is increasing among MRSA isolates. As a result, monitoring and control of aminoglycoside resistance can be effective in the treatment of MRSA isolates. Also, typing of S. aureus isolates based on coagulase gene polymorphism is a suitable method for epidemiological studies.

Aminoglycoside-Modifying Enzymes Are Sufficient to Make Pseudomonas aeruginosa Clinically Resistant to Key Antibiotics.

Aminoglycosides are widely used to treat infections of Pseudomonas aeruginosa. Genes encoding aminoglycoside-modifying enzymes (AMEs), acquired by horizontal gene transfer, are commonly associated with aminoglycoside resistance, but their effects have not been quantified. The aim of this research was to determine the extent to which AMEs increase the antibiotic tolerance of P. aeruginosa. Bioinformatics analysis identified AME-encoding genes in 48 out of 619 clinical isolates of P. aeruginosa, with ant(2′)-Ia and aac(6′)-Ib3, which are associated with tobramcyin and gentamicin resistance, being the most common. These genes and aph(3′)-VIa (amikacin resistance) were deleted from antibiotic-resistant strains. Antibiotic minimum inhibitory concentrations (MICs) were reduced by up to 64-fold, making the mutated bacteria antibiotic-sensitive in several cases. Introduction of the same genes into four antibiotic-susceptible P. aeruginosa strains increased the MIC by up to 128-fold, making the bacteria antibiotic-resistant in all cases. The cloned genes also increased the MIC in mutants lacking the MexXY-OprM efflux pump, which is an important contributor to aminoglycoside resistance, demonstrating that AMEs and this efflux pump act independently in determining levels of aminoglycoside tolerance. Quantification of the effects of AMEs on antibiotic susceptibility demonstrates the large effect that these enzymes have on antibiotic resistance.

Identification of major sequence types among aminoglycoside resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from clinical samples.

Aminoglycosides have been widely used for treating severe staphylococcal infections. Production aminoglycoside modifying enzymes (AMEs) is the main mechanism of resistance to this antibiotic. The aim of this study was to determine the prevalence of AME genes and molecular characterization of aminoglycoside-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from clinical specimens in Iran. A total of 42 clinical isolates of Gram-positive cocci (20 S. aureus and 22 S. epidermidis) with resistance to gentamicin were tested for antimicrobial resistance and differentiated by multilocus sequence typing (MLST). All 42 isolates were resistant to methicillin, kanamycin, and most of them were also resistant to amikacin (98%), tobramycin (98%) and netilmycin (78.5%). Overall, aac(6')-Ie-aph(2")-Ia was the dominant AME gene found in 100% of isolates, followed by aph(3')IIIa found in 90% of isolates. MLST classified S. aureus and S. epidermidis into 5 and 9 distinct sequence types (ST), respectively. The majority of the strains belonged to ST239 (50%) for S. aureus and ST2 (36%) for S. epidermidis. The resistance to aminoglycosides was mainly due to the presence of the aac(6')-Ie-aph(2")-Ia and aph(3') IIIa genes as well as the ST239 for S. aureus and ST2 for S. epidermidis have become the predominant clones in the selected university hospital of Tehran, Iran. Thus, it is critical that clinicians and healthcare workers are aware of the population of S. aureus and S. epidermidis present in order to make decisions for appropriate treatment and infection control practices.

Resistance mechanisms in Gram-negative bacteria

Enterobacterales resistant to carbapenems or producing extended-spectrum β-lactamases (ESBL) and non-fermenters resistant to carbapenems present resistance to many of the antimicrobials commonly used in clinical practice, and have been recognized by the World Health Organization as a critical priority for the development of new antimicrobials. In this review, the main mechanisms of resistance of Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia to β-lactams, quinolones, aminoglycosides and polymyxins will be addressed. Updated information will be presented on the importance in resistance of antimicrobial modification mechanisms (including class C or extended-spectrum β-lactamases, carbapenemases and aminoglycoside-modifying enzymes), permeability alterations due to porin or lipopolysaccharide expression disorders, production of active efflux pumps, target alterations or protection, and expression of two-component systems.

Structure-Activity Relationships for 5'' Modifications of 4,5-Aminoglycoside Antibiotics.

Modification at the 5''-position of 4,5-disubstituted aminoglycoside antibiotics (AGAs) to circumvent inactivation by aminoglycoside modifying enzymes (AMEs) is well known. Such modifications, however, unpredictably impact activity and affect target selectivity thereby hindering drug development. A survey of 5''-modifications of the 4,5-AGAs and the related 5-O-furanosyl apramycin derivatives is presented. In the neomycin and the apralog series, all modifications were well-tolerated, but other 4,5-AGAs require a hydrogen bonding group at the 5''-position for maintenance of antibacterial activity. The 5''-amino modification resulted in parent-like activity, but reduced selectivity against the human cytosolic decoding A site rendering this modification unfavorable in paromomycin, propylamycin, and ribostamycin. Installation of a 5''-formamido group and, to a lesser degree, a 5''-ureido group resulted in parent-like activity without loss of selectivity. These lessons will aid the design of next-generation AGAs capable of circumventing AME action while maintaining high antibacterial activity and target selectivity.

Combined in silico approach and whole genome sequencing: Acinetobacter baumannii ST218 isolate harboring ADC-73 β-lactamase which has a similar C-loop with ADC-56 and ADC-68 β-lactamase

PurposeMultidrug-resistant Acinetobacter baumannii is a noteworthy nosocomial-pathogen and these pathogen-borne infections are difficult to treat. It is significant to make strain typing with WGS and to add new genome data to the literature. Therefore, in our study, we aimed to strain typing of the A. baumannii (A24) isolated from Turkey and reveal informations about ADC-73 β-lactamase. MethodsVITEK 2 system was used for the determination of antibiotic susceptibility. WGS was done on the Illumina NovaSeq 6000 platform. WGS results were analyzed with VFDB, ResFinder, PubMLST, IS Finder. Web-based bioinformatics software, homology modelling, molecular docking and dynamics simulations were used to determine all structural information about ADC-73 β-lactamase. ResultsA24 was found to be multidrug-resistant. Various virulence factors were found in A24. The sequence type of the isolate was determined as ST218. Genes encoding β-lactamase and aminoglycoside modifying enzymes, and IS elements were present in the genome of A24. Besides, secondary and 3D structures of ADC-73 were analyzed. Following, cefepime and imipenem were docked to ADC-56, ADC-68, and ADC-73 and interactions and stability of substrates were simulated. The binding-energies of imipenem to ADC-68 and ADC-73 were calculated −9.44 and −5.98 kcal/mol, respectively. Likewise, binding-energies of cefepime to ADC-56 and ADC-73 were calculated as −19.84 and −36.54 kcal/mol. ConclusionA. baumannii ST218 isolate containing ADC-73 was reported for the first time in Turkey by WGS, and the effect of G225S mutation in this β-lactamase on conformational change and possible interactions with cefepime and impinem were investigated in silico.