Genetic background of aminoglycoside-modifying enzymes in various genetic lineages of clinical aminoglycosides-resistant E. coli and K. pneumoniae isolates in Tunisia.

AIMS: 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. MATERIALS AND 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 ß-lactamase enzymes among genetically heterogeneous clinical isolates present a matter of concern.

Contribution of different mechanisms to aminoglycoside resistance in clinical isolates of Acinetobacter baumannii.

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.

Generating Genotype-Specific Aminoglycoside Combinations with Ceftazidime/Avibactam for KPC-Producing Klebsiella pneumoniae.

Antibiotic combinations, including ceftazidime/avibactam (CAZ/AVI), are frequently employed to combat KPC-producing Klebsiella pneumoniae (KPC-Kp), though such combinations have not been rationally optimized. Clinical KPC-Kp isolates with common genes encoding aminoglycoside-modifying enzymes (AMEs), aac(6')-Ib' or aac(6')-Ib, were used in static time-kill assays (n = 4 isolates) and the hollow-fiber infection model (HFIM; n = 2 isolates) to evaluate the activity of gentamicin, amikacin, and CAZ/AVI alone and in combinations. A short course, one-time aminoglycoside dose was also evaluated. Gentamicin plus CAZ/AVI was then tested in a mouse pneumonia model. Synergy with CAZ/AVI was more common with amikacin for aac(6')-Ib'-containing KPC-Kp but more common with gentamicin for aac(6')-Ib-containing isolates in time-kill assays. In the HFIM, although the isolates were aminoglycoside-susceptible at baseline, aminoglycoside monotherapies displayed variable initial killing, followed by regrowth and resistance emergence. CAZ/AVI combined with amikacin or gentamicin resulted in undetectable counts 50 h sooner than CAZ/AVI monotherapy against KPC-Kp with aac(6')-Ib'. CAZ/AVI monotherapy failed to eradicate KPC-Kp with aac(6')-Ib and a combination with gentamicin led to undetectable counts 70 h sooner than with amikacin. A one-time aminoglycoside dose with CAZ/AVI provided similar killing to aminoglycosides dosed for 7 days. In the mouse pneumonia model (n = 1 isolate), gentamicin and CAZ/AVI achieved a 6.0-log10 CFU/lung reduction at 24 h, which was significantly greater than either monotherapy (P < 0.005). Aminoglycosides in combination with CAZ/AVI were promising for KPC-Kp infections; this was true even for a one-time aminoglycoside dose. Selecting aminoglycosides based on AME genes or susceptibilities can improve the pharmacodynamic activity of the combination.

Rapid and Accurate Detection of Aminoglycoside-Modifying Enzymes and 16S rRNA Methyltransferases by Targeted Liquid Chromatography-Tandem Mass Spectrometry.

New and rapid diagnostic methods are needed for the detection of antimicrobial resistance to aid in curbing drug-resistant infections. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method that could serve this purpose, as it can detect specific peptides of antimicrobial resistance mechanisms with high accuracy. In the current study, we developed an accurate and rapid targeted LC-MS/MS assay based on parallel reaction monitoring for detection of the most prevalent aminoglycoside-modifying enzymes and 16S rRNA methyltransferases in Escherichia coli and Klebsiella pneumoniae that confer resistance to aminoglycosides. Specific tryptic peptides needed for detection were selected and validated for AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, AAC(6')-Ib-cr, ANT(2″)-I, APH(3')-VI, ArmA, RmtB, RmtC, and RmtF. In total, 205 isolates containing different aminoglycoside resistance mechanisms that consisted mostly of E. coli and K. pneumoniae were selected for assay development and evaluation. Mass spectrometry results were automatically analyzed and were compared to whole-genome sequencing results. Of the 2,460 isolate and resistance mechanism combinations tested, 2,416 combinations matched. Discrepancies were further analyzed by repeating LC-MS/MS analysis and performing additional PCRs. Mass spectrometry results were also used to predict resistance and susceptibility to gentamicin, tobramycin, and amikacin in only the E. coli and K. pneumoniae isolates (n = 191). The category interpretations were correctly predicted for gentamicin in 97.4% of the isolates, for tobramycin in 97.4% of the isolates, and for amikacin in 82.7% of the isolates. Targeted LC-MS/MS can be applied for accurate and rapid detection of aminoglycoside resistance mechanisms.

Maldi-TOF MS identification and antibiotic resistance of Escherichia coli isolated from playground.

In this study, it was aimed to determine the antibiotic resistance of Escherichia coli strains isolated from samples taken from various children's parks of Ankara and to confirm the resistance by molecular methods. Five hundred fifty-four samples, including soil samples from 140 different parks and 414 swab samples from slides, swings, ferris wheels, seesaws, and other toys from 176 different parks, were taken. Fourty E. coli strains isolated from these samples were included in the study. Antibiotic susceptibility tests of 40 E. coli isolates were performed by EUCAST recommendations. The resistance rates of E. coli isolates were found as ciprofloxacin 5%, ampicillin 17%, trimethoprim/sulfamethoxazole 15%, streptomycin 12.5%, tobramycin 5%, gentamicin 5%, cefotaxime 2.5%, and ceftazidime 2.5%. Intermediate rates were found as 95%, 90%, and 70% for tobramycin, gentamicin, and streptomycin respectively. blaCTX-M ß-lactamase gene was investigated for an isolate determined to be resistant to both cefotaxime and ceftazidime but blaCTXM gene could not be detected. Aminoglycoside resistance of strains has been investigated because of high intermediate sensitivity rates. For this purpose, aac(6')-Ib, aac(3')-IIa, aph(3')-VI, ant(3')-I, aac(3')-IV, ant(2')-Ia genes scanned, and were detected 97.5% of our isolates ant (3')-I, %25 aac(6')-Ib', 5% aac(3')-IIa, 2.5% ant(2')-Ia. Also, aph(3')-VI, and aac(3')-IV genes could not be detected in any of the isolates. Consequently, it has been revealed that resistant E. coli strains isolated from children's parks can pose a potential risk in public health for transmission of resistant genes.

Unraveling the Gentamicin Drug Product Complexity Reveals Variation in Microbiological Activities and Nephrotoxicity.

The gentamicin drug product is a complex mixture of numerous components, many of which have not individually undergone safety and efficacy assessments. This is in contrast to the majority of medicines that require rigorous characterizations of trace impurities and are dosed as single components. In gentamicin, four components, known as gentamicin congeners C1, C1a, C2, and C2a, comprise the majority of the mixture. A liquid chromatography-mass spectroscopy analysis revealed that the relative abundances of each gentamicin congener in commercial formulations can vary up to 1.9-fold depending on the commercial source of the gentamicin. To determine if the gentamicin used for antibiotic susceptibility testing (AST) would be predictive of the microbiological activity of the product used to dose patients, the relative abundances of the four congeners contained on commercial AST disks were measured. It was found that the congener abundances on the commercial AST disks varied up to 4.1-fold. After purification of the four gentamicin congeners, similar potencies against bacterial strains lacking aminoglycoside-modifying enzymes (AMEs) were observed. However, the potency of the congeners against strains harboring a common AME differed up to 128-fold. Nephrotoxicity of the individual gentamicin congeners also differed significantly in cell-based and repeat-dose rat nephrotoxicity studies. Variations in the composition of commercial gentamicin products combined with toxicity differences between gentamicin congeners suggest that some gentamicin formulations may be more nephrotoxic. Our results also raise the concern that gentamicin susceptibility test results may not be predictive of patient outcomes and could lead to unexpected clinical treatment failures.

Expression of the MexXY Aminoglycoside Efflux Pump and Presence of an Aminoglycoside-Modifying Enzyme in Clinical Pseudomonas aeruginosa Isolates Are Highly Correlated.

The impact of MexXY efflux pump expression on aminoglycoside resistance in clinical Pseudomonas aeruginosa isolates has been debated. In this study, we found that, in general, elevated mexXY gene expression levels in clinical P. aeruginosa isolates confer to slight increases in aminoglycoside MIC levels; however, those levels rarely lead to clinically relevant resistance phenotypes. The main driver of resistance in the clinical isolates studied here was the acquisition of aminoglycoside-modifying enzymes (AMEs). Nevertheless, acquisition of an AME was strongly associated with mexY overexpression. In line with this observation, we demonstrate that the introduction of a gentamicin acetyltransferase confers to full gentamicin resistance levels in a P. aeruginosa type strain only if the MexXY efflux pump was active. We discuss that increased mexXY activity in clinical AME-harboring P. aeruginosa isolates might affect ion fluxes at the bacterial cell membrane and thus might play a role in the establishment of enhanced fitness that extends beyond aminoglycoside resistance.

Detection of antibiotic resistance profiles and aminoglycoside-modifying enzyme (AME) genes in high-level aminoglycoside-resistant (HLAR) enterococci isolated from raw milk and traditional cheeses in Turkey.

The aim of this study was isolation and identification of the high-level aminoglycoside-resistant (HLAR) enterococci in raw milk and dairy products and to analyze their antibiotic resistance and the presence of aminoglycoside-modifying enzyme (AME) genes. A total of 59 HLAR enterococci were isolated from raw milk and traditional cheese samples. Thirty-nine of the 59 HLAR enterococci were isolated on streptomycin-containing agar medium, while the other 20 HLAR strains were isolated on gentamicin containing agar medium. The 59 HLAR enterococci were identified as 26 E. faecalis (44.07%), 18 E. faecium (30.51%), 13 E. durans (22.03%), and two E. gallinarum (3.39%) by species-specific PCR. Disk diffusion tests showed that teicoplanin were the most effective antibiotics used in this study, while 89.83% of isolates were found to be resistant to tetracycline. High rates of multiple antibiotic resistance were detected in HLAR isolates. Minimum inhibitory concentration (MIC) values of HLAR enterococci against streptomycin and gentamicin were found in the range of 64 to > 4096 µg/mL. Forty-seven (79.66%) of the 59 HLAR enterococci were found to be both high-level streptomycin-resistant (HLSR) and high-level gentamicin-resistant (HLGR) by MIC tests. However, no correlation was found between the results of the disk diffusion and MIC tests for gentamicin and streptomycin in some HLAR strains. The aph(3')-IIIa (94.92%) was found to be most prevalent AME gene followed by ant(4')-Ia (45.76%), ant(6')-Ia (20.34%) and aph(2'')-Ic (10.17%). None of the isolates contained the aac(6')-Ie-aph(2'')-Ia, aph(2'')-Ib or aph(2'')-Id genes. None of the AME-encoding genes were identified in E. durans RG20.1, E. faecalis RG22.4, or RG26.1. In conclusion, HLAR enterococci strains isolated in this study may act as reservoirs in the dissemination of antibiotic resistance genes.

Molecular Characterization of Multidrug-Resistant Pseudomonas aeruginosa Isolates in Hospitals in Myanmar.

The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa has become a serious worldwide medical problem. This study was designed to clarify the genetic and epidemiological properties of MDR P. aeruginosa strains isolated from hospitals in Myanmar. Forty-five MDR P. aeruginosa isolates obtained from different patients in seven hospitals in Myanmar were screened using the broth microdilution method. The whole genomes of the MDR isolates were sequenced using a MiSeq platform (Illumina). Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced, and drug resistance genes were identified. Of the 45 isolates, 38 harbored genes encoding carbapenemases, including DIM-1, IMP-1, NDM-1, VIM-2, and VIM-5, and 9 isolates had genes encoding 16S rRNA methylases, including RmtB, RmtD3, RmtE, and RmtF2. Most MDR P. aeruginosa strains isolated in Myanmar belonged to sequence type 1047 (ST1047). This is the first molecular epidemiological analysis of MDR P. aeruginosa clinical isolates in Myanmar. These findings strongly suggest that P. aeruginosa ST1047 strains harboring carbapenemases, including DIM-, IMP-, NDM-, and VIM-type metallo-ß-lactamases, have been spreading throughout medical settings in Myanmar.

Coexistence of genes encoding aminoglycoside modifying enzymes among clinical Acinetobacter baumannii isolates in Ahvaz, Southwest Iran.

Aminoglycosides are widely recommended for treatment of Acinetobacter baumannii infections in combination with ß-lactams or quinolones. This cross-sectional study was aimed to investigate the coexistence of aminoglycoside modifying enzyme (AME) genes among A. baumannii isolates from clinical samples in Ahvaz, Iran. A total of 85 clinical A. baumannii isolates typed by ERIC-PCR were investigated for the presence of AME genes, including ant(3″)-Ia, aac(6')-Ib, aac(3')-Ia, ant(2″)-Ia, and aph(3')-VIa by PCR. The resistance rates to aminoglycoside agents were evaluated by disk diffusion. In this study, 84 out of 85 A. baumannii isolates were resistant to at least one of the aminoglycosides and harbored at least one AME gene. The most common gene encoding AMEs was aph (3')VIa, followed by aac(3')-Ia, ant(3″)-Ia, ant (2″)-Ia, and aac(6')-Ib. The aminoglycoside-resistant genotypes were completely matched to resistant phenotypes to each one of the aminoglycoside agents. There was a clear association between AME gene types and the phenotype of resistance to aminoglycosides with their ERIC-PCR types. Our findings highlight the coexistence of AME genes and clonal dissemination of multiresistant A. baumannii in hospital setting.

Occurrence of genes encoding aminoglycoside-modifying enzymes in Escherichia coli isolates from chicken meat.

1. The aim of the experiment was to determine the occurrence of genes encoding aminoglycoside-modifying enzymes (AMEs) in Escherichia coli isolates recovered from chicken meat.2. Antibiotic sensitivity was tested using the disc diffusion test. AMEs and virulence profile were determined by PCR/sequencing.3. Out of 195 meat samples collected, 185 (95%) isolates were identified as E. coli. Disc diffusion showed a resistance value of 22% (n = 42) for at least one of the antibiotic aminoglycosides (AGs) tested (tobramycin, gentamycin, amikacin and kanamycin). PCR screening showed the presence of three classes of AMEs, namely, aac(3)-II (12%), aac(6')-Ib (7%) and aac(2')-Ia (5%). Eight of the 42 isolates were positive for the stx1 and sxt2 genes and were defined as Shiga toxin-producing E coli., while the eae gene was positive in one strain. Among the 42 isolates, group A was the predominant phylogenetic identified (76%), followed by group D (21%). One isolate belonged to subgroup B23.4. The results suggested that chicken meat could be an important reservoir of AMEs, and pose a potential risk by dissemination of resistance to humans through the food chain.

Whole-Genome Sequence Analysis of Multidrug-Resistant Campylobacter Isolates: a Focus on Aminoglycoside Resistance Determinants.

A whole-genome sequencing (WGS) approach was conducted in order to identify the molecular determinants associated with antimicrobial resistance in 12 multidrug-resistant Campylobacter jejuni and Campylobacter coli isolates, with a focus on aminoglycoside resistance determinants. Two variants of a new aminoglycoside phosphotransferase gene [aph(2″)-Ii1 and aph(2″)-Ii2 ] putatively associated with gentamicin resistance were found. In addition, the following new genes were identified for the first time in Campylobacter: a lincosamide nucleotidyltransferase gene [lnu(G)], likely associated with lincomycin resistance, and two resistance enzyme genes (spw and apmA) similar to those found in Staphylococcus aureus, which may confer spectinomycin and gentamicin resistance, respectively. A C1192T mutation of the 16S rRNA gene that may be involved in spectinomycin resistance was also found in a C. coli isolate. Genes identified in the present study were located either on the bacterial chromosome or on plasmids that could be transferred naturally. Their role in aminoglycoside resistance remains to be supported by genetic studies. Regarding the other antimicrobial agents studied, i.e., ampicillin, ciprofloxacin, erythromycin, and tetracycline, a perfect correlation between antimicrobial phenotypes and genotypes was found. Overall, our data suggest that WGS analysis is a powerful tool for identifying resistance determinants in Campylobacter and can disclose the full genetic elements associated with resistance, including antimicrobial compounds not tested routinely in antimicrobial susceptibility testing.

Aminoglycoside binding and catalysis specificity of aminoglycoside 2″-phosphotransferase IVa: A thermodynamic, structural and kinetic study.

BACKGROUND: Aminoglycoside O-phosphotransferases make up a large class of bacterial enzymes that is widely distributed among pathogens and confer a high resistance to several clinically used aminoglycoside antibiotics. Aminoglycoside 2″-phosphotransferase IVa, APH(2″)-IVa, is an important member of this class, but there is little information on the thermodynamics of aminoglycoside binding and on the nature of its rate-limiting step. METHODS: We used isothermal titration calorimetry, electrostatic potential calculations, molecular dynamics simulations and X-ray crystallography to study the interactions between the enzyme and different aminoglycosides. We determined the rate-limiting step of the reaction by the means of transient kinetic measurements. RESULTS: For the first time, Kd values were determined directly for APH(2″)-IVa and different aminoglycosides. The affinity of the enzyme seems to anti-correlate with the molecular weight of the ligand, suggesting a limited degree of freedom in the binding site. The main interactions are electrostatic bonds between the positively charged amino groups of aminoglycosides and Glu or Asp residues of APH. In spite of the significantly different ratio Kd/Km, there is no large difference in the transient kinetics obtained with the different aminoglycosides. We show that a product release step is rate-limiting for the overall reaction. CONCLUSIONS: APH(2″)-IVa has a higher affinity for aminoglycosides carrying an amino group in 2' and 6', but tighter bindings do not correlate with higher catalytic efficiencies. As with APH(3')-IIIa, an intermediate containing product is preponderant during the steady state. GENERAL SIGNIFICANCE: This intermediate may constitute a good target for future drug design.

Comprehensive study to investigate the role of various aminoglycoside resistance mechanisms in clinical isolates of Acinetobacter baumannii.

Therapeutic resistance towards most of the current treatment regime by Acinetobacter baumannii has reduced the prescribing antibiotic pattern and option is being re-shifted towards more toxic agents including aminoglycosides. The present investigation aimed at to study various mechanisms towards aminoglycoside non-susceptibility in clinical isolates of A. baumannii. The bacteria were subjected to genetic basis assessment for the presence of aminoglycoside modifying enzymes (AME), 16S rRNA methylase encoding genes and relative expression of AdeABC and AbeM efflux pumps in relation to their susceptibility to five aminoglycosides. When isolates were subjected to typing by repetitive extragenic palindromic (REP) PCR, isolates could be separated into thirteen definite clones. The majority of isolates (94%) were positive for AME encoding genes. Possession of ant(2')-Ia correlated with non-susceptibility towards gentamicin, amikacin, kanamycin, tobramycin; while, presence of aph(3')-VIa attributed to resistance towards amikacin, kanamycin; possession of aac(3')-Ia allied with non-susceptibility to amikacin, tobramycin and presence of aac(3')IIa correlated with kanamycin non-susceptibility. Presence of armA was detected in 34.4%, 34.2%, 29.2%, 40.3%, and 64.2% of isolates showing non-susceptibility to gentamicin, amikacin, kanamycin, tobramycin and netilmicin, respectively. No isolates were found to carry rmtB or rmtC. Amikacin non-susceptibility in comparison to other aminoglycosides correlated with over production of adeB. Overall, the results represented a definitive correlation between presence of AME encoding genes as well as armA and resistance of A. baumannii towards aminoglycosides. On the other hand, the up-regulation of AdeABC and AbeM systems was found to have only the partial role in development of aminoglycoside resistance.

Amikacin: Uses, Resistance, and Prospects for Inhibition.

Aminoglycosides are a group of antibiotics used since the 1940s to primarily treat a broad spectrum of bacterial infections. The primary resistance mechanism against these antibiotics is enzymatic modification by aminoglycoside-modifying enzymes that are divided into acetyl-transferases, phosphotransferases, and nucleotidyltransferases. To overcome this problem, new semisynthetic aminoglycosides were developed in the 70s. The most widely used semisynthetic aminoglycoside is amikacin, which is refractory to most aminoglycoside modifying enzymes. Amikacin was synthesized by acylation with the l-(-)-γ-amino-α-hydroxybutyryl side chain at the C-1 amino group of the deoxystreptamine moiety of kanamycin A. The main amikacin resistance mechanism found in the clinics is acetylation by the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib], an enzyme coded for by a gene found in integrons, transposons, plasmids, and chromosomes of Gram-negative bacteria. Numerous efforts are focused on finding strategies to neutralize the action of AAC(6')-Ib and extend the useful life of amikacin. Small molecules as well as complexes ionophore-Zn+2 or Cu+2 were found to inhibit the acetylation reaction and induced phenotypic conversion to susceptibility in bacteria harboring the aac(6')-Ib gene. A new semisynthetic aminoglycoside, plazomicin, is in advance stage of development and will contribute to renewed interest in this kind of antibiotics.

The prevalence of aminoglycoside-modifying enzymes among coagulase negative staphylococci in Iranian pediatric patients.

In spite of widespread emergence of aminoglycoside resistance, these drugs are still used in the treatment of staphylococcal infections. This study aimed to investigate the distribution of aminoglycoside resistance and genes encoding aminoglycoside - modifying enzymes (AMEs) as well as Staphylococcal Cassette Chromosome mec (SCCmec) type in coagulase negative staphylococci (CoNS) in pediatric patients. Totally, 93 CoNS isolates were examined for susceptibility to aminoglycosides using disk diffusion and/or E-test methods. AMEs genes and SCCmec types were detected using multiplex PCR. Strain typing was performed using repetitive extragenic palindromic (REP) - PCR assay. The non-susceptibility rates to kanamycin, tobramycin, gentamicin, amikacin and netilmicin were 73%, 59%, 49.5%, 16% and 7.5%, respectively. aac(6')-Ie-aph(2″)-Ia, ant(4')-Ia and aph(3')-IIIa were encountered in 56 (60.2%), 38 (40.8%) and 18 (19.3%) isolates, respectively. In aac(6')-Ie-aph(2″)-Ia- positive isolates, the non- susceptibility rates to kanamycin, gentamicin, tobramycin, amikacin and netilmicin were 83%, 74%, 73%, 49% and 43%, respectively. SCCmec types included type IV (n = 31), I (n = 17), II (n = 5), III (n = 4), and V (n = 2). Three isolates had two types; I + III (n = 2) and III + IV (n = 1) whereas 11 isolates were non-typeable. AMEs genes carriers were distributed frequently into type IV. We found diverse fingerprint patterns among our isolates. In conclusion, there was a strong correlation between alarming rate of aminoglycoside resistance and methicillin resistance. Discordances between phenotypic and genotypic detection of aminoglycoside resistance were discernible. AMEs genes might be related to SCCmec types.

Phenotypic and genotypic evaluation of aminoglycoside resistance in Escherichia coli isolated from patients with blood stream infections in Tehran, Iran.

Background and Objectives: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

Structure-Activity Relationship of Pyrrolidine Pentamine Derivatives as Inhibitors of the Aminoglycoside 6'-N-Acetyltransferase Type Ib.

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.

Detection of Aminoglycoside Modifying Enzyme (AME) genes in Acinetobacter baumannii isolates and the inhibitory effect of efflux pump activity on drug susceptibility pattern.

INTRODUCTION: The development of aminoglycoside modifying enzymes (AMEs) and increased efflux activity are considered important aminoglycosides resistance mechanisms. AIM: This study is focused on the detection of the AMEs gene and assessing the effect of efflux pump inhibitor on the reversal of A. baumannii drug susceptibility. METHODOLOGY: Bacterial DNA was amplified using AMEs gene-specific primers. Isolates were also investigated for efflux pump activity using efflux pump inhibitor (EPI) i.e. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and the impact of both mechanisms was analyzed. RESULTS: Among A. baumannii isolates, 55% isolates (n â€‹= â€‹22/40) were identified to have aminoglycoside modifying enzymes genes; ant(3')-I gene (50%, 11/22), aac(6')-Ib gene (45.4%, 10/22), aph(3')-I gene (18.1%, 4/22) and aac(3)-I (9.1%, 2/22). Total 70% isolates have shown MIC alteration in different classes of drugs in response to EPI-CCCP. Such alteration was found in 100% amikacin sensitive and 58.6% amikacin resistant, 93.7% and 57.1% gentamicin sensitive and resistant isolates respectively. CONCLUSION: The presence of aminoglycosides modifying enzymes was frequent among aminoglycosides resistant A. baumannii isolates and the coexistence of efflux pumps activity also plays an important role to increase drug resistance. REPOSITORIES: Genbank and their accession numbers are MT903331[aac(3)-I], MT903332 MT903333 [ant(3')-I], MT903334, MT903335 [aph(3')-I)] and MT903336, MT940242 [ aac(6')-Ib].

Inhibitors of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] identified by in silico molecular docking.

AAC(6')-Ib is an important aminoglycoside resistance enzyme to target with enzymatic inhibitors. An in silico screening approach was used to identify potential inhibitors from the ChemBridge library. Several compounds were identified, of which two of them, 4-[(2-{[1-(3-methylphenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid and 2-{5-[(4,6-dioxo-1,3-diphenyl-2-thioxotetrahydro-5(2H)-pyrimidinylidene)methyl]-2-furyl}benzoic acid, showed micromolar activity in inhibiting acetylation of kanamycin A. These compounds are predicted to bind the aminoglycoside binding site of AAC(6')-Ib and exhibited competitive inhibition against kanamycin A.