Aminoglycoside-modifying Enzymes Research Articles

Stenotrophomonas maltophilia complex: insights into evolutionary relationships, global distribution and pathogenicity.

Stenotrophomonas maltophilia complex (Smc) comprises opportunistic Gram-negative bacilli responsible for various nosocomial infections. Limited data exists concerning its evolutionary lineage, global prevalence and pathogenicity. We conducted an extensive genomic analysis on 734 Smc genomes, of which 90 were newly sequenced and isolated from different patients. The species composition and evolutionary relationships of Smc were examined using core protein sequence analysis. Pathogenicity evaluation was used by assays for swimming motility, biofilm formation and identification of virulence factors. The broth microdilution method was used to evaluate the drug resistance spectrum of clinical isolates. Phylogenetic analyses delineated 24 species-level clades, dominated by S. maltophilia (42.8%), S. sepilia (13.6%) and S. geniculata (9.9%). Geographically, strains were primarily distributed in Europe (34.2%), Asia (33.7%) and North America (24.0%), with intricate global distribution patterns. Meanwhile, 154 virulence-associated genes and 46 antimicrobial resistance genes within Smc were identified. These genes encoded span various functions, including motility, adherence, toxin, RND antibiotic efflux pumps, beta-lactamases and aminoglycoside-modifying enzymes. Moreover, significant variations were indicated in swimming motility and biofilm-forming capability across the different species, with S. sepilia exhibiting superior levels of both traits. Additionally, no statistically significant discrepancy was detected among Smc species to other antibiotics, despite the fact that all S. geniculata isolates were resistant to Ceftazidime and much higher than other species. Our findings indicate the need to pay increased attention to other mainstream species of Smc besides S. maltophilia in order to better manage Smc-related infections and tailor effective treatment strategies.

Dissemination and Molecular Characterization of 16s Rna Methylase and Aminoglycoside Modifying Enzymes- Producing P. Aeruginosa Isolated from Burn Specimens in Najaf City

Background: Pseudomonas aeruginosa is a significant species of bacteria connected with nosocomial infections involving, pneumonia, infections of urinary tract, and damage of the surgical site. Aim of the Study: The aim of this thesis is to investigate the occurrence of genes encoding clinically important 16S rRNA methylases and AMEs enzymes in aminoglycosides resistance P.aeruginosa isolates recovered from Najaf hospitals and the characteristics of these isolates. Methods: a total of 68 specimens were isolated from patients at burn center in Najaf City from the whole study period. The 24 isolates were screened for the sensitivity versus 3 various antibiotics disks of the one class by using Kirby-Bauer disk diffusion method. Results: The resistance rate of 24 isolates of XDR Pseudomonas aeruginosa against aminoglycoside groups: gentamicin, amikacin, and tobramycin were 91.6 %, 83.3 %, and 66.6 %, respectively. However, the resistance profiles of the isolates show that 22 (91.6 %) were resistant to 1 or more antibiotics of aminoglycoside. Present study show that only 3 (12.5%) of XDR isolates harbored ant(4')-IIb gene isolates and none of the XDR isolates investigated were positive for rmtA and aac(6')-IIb gene. Conclusions: This survey has showed for the initial time three isolates harbors the ant(4')-IIb aminoglycoside resistance genes in Iraq.

Resistance to aminoglycoside and quinolone drugs among Klebsiella pneumoniae clinical isolates from northern Jordan

This study aimed to identify phenotypic and genotypic aminoglycoside and quinolone non-susceptibility and the prevalence of aminoglycoside-modifying enzymes and plasmid-mediated quinolone resistance genes among K. pneumoniae clinical isolates from northern Jordan. K. pneumoniae isolates (n = 183) were tested for antimicrobial susceptibility using the Kirby-Bauer disk diffusion method. The double-disk synergy test was used for the detection of the extended-spectrum beta-lactamase phenotype. Polymerase chain reaction was used to detect genes encoding aminoglycoside-modifying enzyme (aac (3′)-II, aac (6′)-II, aac (6′)-Ib, ant (3″)-I, aph (3′)-VI, armA, and rmtB), and plasmid-mediated quinolone resistance (qnrA, qnrB, qnrC, qnrD, qnrS, acc(6′)-Ib–cr, qepA, and oqxAB) genes. Multi-locus sequence typing was used to elucidate the genetic diversity of selected isolates. The non-susceptibility percentages to aminoglycosides and quinolones were 65.0 % and 61.7 %, respectively. The most frequent aminoglycoside-modifying enzyme gene was ant (3″)-I at 73.8 %, followed by aac (6′)-Ib at 25.1 %, aac (3′)-II at 17.5 %, aph (3′)-VI at 12.0 %, armA at 9.8 %, and rmtB at 0.5 %. Aac (6′)-II was not detected among the isolates. The most frequent plasmid-mediated quinolone resistance gene was oqxAB at 31.7 %, followed by qnrS at 26.2 %, qnrB at 25.7 %, and aac(6′)-Ib–cr at 25.7 %. QnrA, qnrD, qebA, and qnrC were not detected among the isolates. Aac (3′)-II, aac (6′)-Ib, aph (3′)-VI, armA, qnrB, qnrS, and acc(6′)-Ib–cr were significantly associated with non-susceptibility to aminoglycosides, quinolones, and beta-lactams. Among 27 randomly selected K. pneumoniae isolates, the most common sequence type was ST2096, followed by ST348 and ST1207. Overall, 19 sequence types were observed, confirming a high level of genetic diversity among the isolates. High percentages of non-susceptibility to the studied antimicrobials were found and were associated with the presence of several resistance genes. Similar studies should be periodically carried out to monitor changes in the prevalence of resistance phenotypes and genotypes of isolates.

First Report of aac(6')-Ib and aac(6')-Ib-cr Variant Genes Associated with Mutations in gyrA Encoded Fluoroquinolone Resistance in Avian Campylobacter coli Strains Collected in Tunisia.

The aac(6')-Ib gene is the most widespread gene encoding aminoglycoside-modifying enzyme and conferring resistance to tobramycin, streptomycin and kanamycin. The variant aac(6')-Ib-cr gene confers resistance to both aminoglycosides and fluoroquinolones (FQ). A total of 132 Campylobacter isolates, including 91 C. jejuni and 41 C. coli, were selected from broiler hens isolates. The aac(6')-Ib gene was amplified using PCR and was subsequently digested with the BtsCI restriction enzyme to identify aac(6')-Ib-cr. Among these isolates, 31 out of 41 C. coli (75.6%) and 1 (0.98%) C. jejuni were positive for the aac(6')-Ib gene, which was identified as the aac(6')-Ib-cr variant in 10 (32.25%) C. coli isolates. This variant was correlated with mutations in gyrA (Thr-86-Ile), as well as resistance to FQs. This study is the first report in Tunisia on Campylobacter coli strains harboring both the aac(6')-Ib and aac(6')-Ib-cr variants. These genes were present in Campylobacter isolates exhibiting resistance to multiple antibiotics, which restricts the range of available treatments.

Extended spectrum beta-lactamase and aminoglycoside modifying enzyme genes in multi drug resistant Gram-negative bacteria: A snapshot from a tertiary care centre

Extended spectrum beta-lactamase and aminoglycoside modifying enzyme genes in multi drug resistant Gram-negative bacteria: A snapshot from a tertiary care centre

Serratia marcescens enzyme SME-2 isolated from sputum in New Zealand.

The Serratia marcescens enzymes (SMEs) are chromosomally encoded Ambler Class A carbapenem-hydrolysing β-lactamases, which distinctively express resistance to carbapenems while remaining susceptible to extended-spectrum cephalosporins. Global reports of SMEs are infrequent. Here we describe the isolation of an SME-2-producing S. marcescens from the sputum of a patient who was hospitalized at Christchurch Hospital, New Zealand. An immunosuppressed asthmatic patient who presented with shortness of breath and hypoxia grew S. marcescens from a sputum culture. Antimicrobial susceptibilities were determined by Phoenix, with MICs of meropenem and imipenem determined by Liofilchem® MIC gradient strips and interpreted according to EUCAST breakpoints. Investigation for carbapenemase was performed using Carba NP, modified CIM (mCIM) and GeneXpert® Carba-R. WGS was performed using the Illumina DNA Prep library kit and sequenced using MiSeq. The isolate showed an unusual susceptibility profile, including high-level resistance to meropenem and imipenem, while remaining susceptible to extended-spectrum cephalosporins. The Carba NP and mCIM were positive and WGS demonstrated the presence of a blaSME-2 gene located on the chromosome within the SmarGI1-1 genomic island. In addition, a blaSRT-like class C β-lactamase, aac(6')-Ic aminoglycoside-modifying enzyme and various multidrug efflux mechanisms were found. Phylogenetic core-genome analysis indicated no matching genome with RefSeq database strains. S. marcescens is an opportunistic pathogen of concern, harbouring a variety of intrinsic resistance mechanisms, including the potential for stable AmpC hyperproduction. Globally, SME-type carbapenemases have been infrequently reported; however, isolates carrying this mechanism could have limited treated options, having implications for patient management. To the best of our knowledge this is the first report of SME in New Zealand.

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

IntroductionThe 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. MethodologyBacterial 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. ResultsAmong A. baumannii isolates, 55% isolates (n ​= ​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. ConclusionThe 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. RepositoriesGenbank 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].

Molecular Typing of Multidrug-Resistant Acinetobacter baumannii Isolates from Clinical Specimens by ERIC-PCR and MLVA.

Acinetobacter baumannii, a Gram-negative and oxidase-negative bacterium, is a major cause of nosocomial infections, leading to high mortality rates in hospitalized patients. The use of 2 prominent molecular typing methods (i.e., enterobacterial repetitive intergenic consensus-polymerase chain reaction [ERIC-PCR] and multiple-locus variable-number tandem repeat [VNTR] analysis [MLVA]) for genotyping A. baumannii isolates has proven to be an effective approach in assessing the clonal relation of these isolates and managing their outbreaks. A total of 100 A. baumannii isolates were collected from immunocompromised patients hospitalized in the intensive care unit (ICU) of a hospital in Zanjan City, Iran. Their antibiotic resistance ability (especially aminoglycoside resistance) was studied by disc diffusion tests. The genetic typing of A. baumannii was studied using ERIC-PCR and MLVA methods. All isolates were resistant to 3 or more antibiotics and regarded as multidrug-resistant (MDR). Additionally, 32% of the isolates were resistant to all antibiotics tested, and 91% were extensively drug-resistant (XDR). The increased rate of aminoglycoside-resistant A. baumannii in ICU patients, with an increased incidence of aminoglycoside-modifying enzymes of aac (6')-Ib, ant (3″)-I, and aph (2″)-Id. ERIC-PCR has likewise shown an increased level of diversity in A. baumannii isolates. According to the ERIC-PCR patterns, isolates were classified as 4 clusters, while according to the MLVA patterns, isolates were classified as 9 distinct clusters. ERIC-PCR and MLVA assays serve as useful genotyping methods to assess the genetic variety or clonal relatedness of A. baumannii isolates.

Prevalence of Aminoglycoside Resistance Genes in Clinical Isolates of Pseudomonas aeruginosa from Taif, Saudi Arabia-An Emergence Indicative Study.

Hospital-acquired infections caused by P. aeruginosa contribute to global distress because of the elevated rates of microbial antibiotic resistance. Aminoglycosides are antipseudomonal agents that are effectively and frequently utilized to eradicate this infection. This current study is a retrospective study investigating plasmid-mediated aminoglycoside resistance by focusing on the prevalence of the genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylase among P. aeruginosa clinical isolates from Taif, Saudi Arabia. A hundred clinical isolates of P. aeruginosa were collected. The isolates were identified from February 2021 to February 2022. Antibiotic susceptibility testing and MICs were determined using (DD) and (BM-MIC) testing, respectively. AMEs and 16S rRNA methylase variants in bacterial isolates were amplified via PCR for genetic detection. A relatively high multiple antibiotic resistance rate corresponding to 10-32% was reported. Eighteen percent of P. aeruginosa isolates were gentamicin-amikacin-tobramycin resistant according to the MIC levels. The aminoglycoside-resistant strains were additionally identified via GyrA gene sequencing. The phylogenic relatedness dendrogram of the sequenced GyrA genes was performed using a neighbor-joining method via MEGAX software version 10.2.6. The most prevalent AME encoding gene was aac(6')-Ib, observed in 94.4% of resistant isolates, while a resistance gene cocktail of [aac(6')-Ib and ant(3″)-I] was a highly frequent combination (27.8%). This study updated the knowledge about aminoglycoside resistance mechanisms in P. aeruginosa, which constitutes an urgent need, especially after the COVID-19 crisis, which was associated with increased antimicrobial use and resistance rates.

Identification and characterization of a novel chromosomal aminoglycoside 3'-O-phosphotransferase, APH(3')-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farm.

Aminoglycosides, as important clinical antimicrobials, are used as second-line drugs for treating multidrug-resistant tuberculosis or combined with β-lactam drugs for treating severe infections such as sepsis. Aminoglycoside-modifying enzyme (AME) is the most important mechanism of aminoglycoside resistance and deserves more attention. The bacterium Kluyvera intermedia DW18 was isolated from the sewage of an animal farm using the conventional method. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of antimicrobials. A novel resistance gene was cloned, and the enzyme was expressed. The kinetic parameters were measured by a SpectraMax M5 multifunctional microplate reader. Bioinformatic analysis was performed to reveal the genetic context of the aph(3')-Id gene and its phylogenetic relationship with other AMEs. A novel aminoglycoside 3'-O-phosphotransferase gene designated aph(3')-Id was identified in K. intermedia DW18 and shared the highest amino acid identity of 77.49% with the functionally characterized aminoglycoside 3'-O-phosphotransferase APH(3')-Ia. The recombinant plasmid carrying the novel resistance gene (pMD19-aph(3')-Id/E. coli DH5α) showed 1,024-, 512-, 128- and 16-fold increased MIC levels for kanamycin, ribostamycin, paromomycin and neomycin, respectively, compared with the reference strain DH5α. APH(3')-Id showed the highest catalytic efficiency for ribostamycin [kcat/Km of (4.96 ± 1.63) × 105 M-1/s-1], followed by paromomycin [kcat/Km of (2.18 ± 0.21) × 105 M-1/s-1], neomycin [kcat/Km of (1.73 ± 0.20) × 105 M-1/s-1], and kanamycin [kcat/Km of (1.10 ± 0.18) × 105 M-1/s-1]. Three conserved functional domains of the aminoglycoside phosphotransferase family and ten amino acid residues responsible for the phosphorylation of kanamycin were found in the amino acid sequence of APH(3')-Id. No mobile genetic element (MGE) was discovered surrounding the aph(3')-Id gene. In this work, a novel aminoglycoside 3'-O-phosphotransferase gene designated aph(3')-Id encoded in the chromosome of the environmental isolate Kluyvera intermedia DW18 was identified and characterized. These findings will help clinicians select effective antimicrobials to treat infections caused by pathogens with this kind of resistance gene.

Extensive screening reveals previously undiscovered aminoglycoside resistance genes in human pathogens

Antibiotic resistance is a growing threat to human health, caused in part by pathogens accumulating antibiotic resistance genes (ARGs) through horizontal gene transfer. New ARGs are typically not recognized until they have become widely disseminated, which limits our ability to reduce their spread. In this study, we use large-scale computational screening of bacterial genomes to identify previously undiscovered mobile ARGs in pathogens. From ~1 million genomes, we predict 1,071,815 genes encoding 34,053 unique aminoglycoside-modifying enzymes (AMEs). These cluster into 7,612 families (<70% amino acid identity) of which 88 are previously described. Fifty new AME families are associated with mobile genetic elements and pathogenic hosts. From these, 24 of 28 experimentally tested AMEs confer resistance to aminoglycoside(s) in Escherichia coli, with 17 providing resistance above clinical breakpoints. This study greatly expands the range of clinically relevant aminoglycoside resistance determinants and demonstrates that computational methods enable early discovery of potentially emerging ARGs.

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.

A survey of frequency of virulence and aminoglycoside antibiotic-resistant genotypes and phenotypes in Escherichia coli in broilers in Khartoum State, Sudan.

Although Escherichia coli (E. coli) is considered a normal microflora in the poultry intestine, certain strains namely, Avian Pathogenic E. coli (APEC), cause colisepticaemia (fatal disease) in poultry. The aim of this study was to determine the prevalence of the virulence genes, i.e. (iroN, ompT, iss, iutA, and hlyF) and aminoglycoside-modifying enzyme (AME) genes, i.e. (strA and strB) in Escherichia coli strains in broilers in Khartoum State. A total of 25 E. coli isolates were collected from broilers farms. All isolates were screened for antimicrobial susceptibility tests using Kirby-Bauer disc diffusion method. In addition, all isolates were tested for the presence of virulence genes and modifying enzyme genes using the polymerase chain reaction (PCR). The results showed that the prevalence of positive strains to virulence genes were 14 (56%), 21 (84%), 14 (56%), 0 (0%) and 0 (0%) to iroN, iutA, hlyF, ompT and iss, respectively. Combined virulence genes include iroN, hlyF and iutA were detected in 14 (56%). The rates of resistance were as follows: Gentamycin: (32%), Kanamycin: (20%) and Streptomycin (16%). Of the genes tested, strA (72%) was the most commonly recognized gene followed by strB (56%). It could be concluded that this is the first report of molecular survey of virulence and aminoglycoside-modifying enzyme (AME) resistant genes in APEC isolates from broiler in Sudan. Therefore, prohibition of non-curative application of antibiotic, dishearten their abuse and to be frequently observant by suppling suitable research-based policy for the poultry industry is warranted.

Staphylococcus aureus Small-Colony Variants from Airways of Adult Cystic Fibrosis Patients as Precursors of Adaptive Antibiotic-Resistant Mutations.

Prototypic Staphylococcus aureus and their small-colony variants (SCVs) are predominant in cystic fibrosis (CF), but the interdependence of these phenotypes is poorly understood. We characterized S. aureus isolates from adult CF patients over several years. Of 18 S. aureus-positive patients (58%), 13 (72%) were positive for SCVs. Characterization included genotyping, SCCmec types, auxotrophy, biofilm production, antibiotic susceptibilities and tolerance, and resistance acquisition rates. Whole-genome sequencing revealed that several patients were colonized with prototypical and SCV-related clones. Some clonal pairs showed acquisition of aminoglycoside resistance that was not explained by aminoglycoside-modifying enzymes, suggesting a mutation-based process. The characteristics of SCVs that could play a role in resistance acquisition were thus investigated further. For instance, SCV isolates produced more biofilm (p < 0.05) and showed a higher survival rate upon exposure to ciprofloxacin and vancomycin compared to their prototypic associated clones. SCVs also developed spontaneous rifampicin resistance mutations at a higher frequency. Accordingly, a laboratory-derived SCV (ΔhemB) acquired resistance to ciprofloxacin and gentamicin faster than its parent counterpart after serial passages in the presence of sub-inhibitory concentrations of antibiotics. These results suggest a role for SCVs in the establishment of persistent antibiotic-resistant clones in adult CF patients.

Genomics of Aminoglycoside Resistance in Pseudomonas aeruginosa Bloodstream Infections at a United States Academic Hospital.

Pseudomonas aeruginosa frequently becomes resistant to aminoglycosides by the acquisition of aminoglycoside modifying enzyme (AME) genes and the occurrence of mutations in the mexZ, fusA1, parRS, and armZ genes. We examined resistance to aminoglycosides in a collection of 227 P. aeruginosa bloodstream isolates collected over 2 decades from a single United States academic medical institution. Resistance rates of tobramycin and amikacin were relatively stable over this time, while the resistance rates of gentamicin were somewhat more variable. For comparison, we examined resistance rates to piperacillin-tazobactam, cefepime, meropenem, ciprofloxacin, and colistin. Resistance rates to the first four antibiotics were also stable, although uniformly higher for ciprofloxacin. Colistin resistance rates were initially quite low, rose substantially, and then began to decrease at the end of the study. Clinically relevant AME genes were identified in 14% of isolates, and mutations predicted to cause resistance were relatively common in the mexZ and armZ genes. In a regression analysis, resistance to gentamicin was associated with the presence of at least one gentamicin-active AME gene and significant mutations in mexZ, parS, and fusA1. Resistance to tobramycin was associated with the presence of at least one tobramycin-active AME gene. An extensively drug-resistant strain, PS1871, was examined further and found to contain five AME genes, most of which were within clusters of antibiotic resistance genes embedded in transposable elements. These findings demonstrate the relative contributions of aminoglycoside resistance determinants to P. aeruginosa susceptibilities at a United States medical center. IMPORTANCE Pseudomonas aeruginosa is frequently resistant to multiple antibiotics, including aminoglycosides. The rates of resistance to aminoglycosides in bloodstream isolates collected over 2 decades at a United States hospital remained constant, suggesting that antibiotic stewardship programs may be effective in countering an increase in resistance. Mutations in the mexZ, fusA1, parR, pasS, and armZ genes were more common than acquisition of genes encoding aminoglycoside modifying enzymes. The whole-genome sequence of an extensively drug resistant isolate indicates that resistance mechanisms can accumulate in a single strain. Together, these results suggest that aminoglycoside resistance in P. aeruginosa remains problematic and confirm known resistance mechanisms that can be targeted for the development of novel therapeutics.

N-Formimidoylation/-iminoacetylation modification in aminoglycosides requires FAD-dependent and ligand-protein NOS bridge dual chemistry

Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of which the allosteric redox switch formed by the lysine-cysteine NOS bridge is an example. Here, we report a noncanonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin to form the antibiotic BD-12. X-ray crystallography was used to investigate this complex enzymatic process, which showed Orf1 has two substrate-binding sites that sit 13.5 Å apart unlike canonical FAD-dependent oxidoreductases. One site could accommodate glycine and the other glycinothricin or glycylthricin. Moreover, an intermediate-enzyme adduct with a NOS-covalent linkage was observed in the later site, where it acts as a two-scissile-bond linkage facilitating nucleophilic addition and cofactor-free decarboxylation. The chain length of nucleophilic acceptors vies with bond cleavage sites at either N–O or O–S accounting for N-formimidoylation or N-iminoacetylation. The resultant product is no longer sensitive to aminoglycoside-modifying enzymes, a strategy that antibiotic-producing species employ to counter drug resistance in competing species.

ANT(9)-Ic, a Novel Chromosomally Encoded Aminoglycoside Nucleotidyltransferase from Brucella intermedia.

Aminoglycoside-modifying enzymes are among the most important mechanisms of resistance to aminoglycoside antibiotics, typically conferring high-level resistance by enzymatic drug inactivation. Previously, we isolated a multidrug-resistant Brucella intermedia strain ZJ499 from a cancer patient, and whole-genome sequencing revealed several putative novel aminoglycoside-modifying enzyme genes in this strain. Here, we report the characterization of one of them that encodes an intrinsic, chromosomal aminoglycoside nucleotidyltransferase designated ANT(9)-Ic, which shares only 33.05% to 47.44% amino acid identity with the most closely related ANT(9)-I enzymes. When expressed in Escherichia coli, ANT(9)-Ic conferred resistance only to spectinomycin and not to any other aminoglycosides tested, indicating a substrate profile typical of ANT(9)-I enzymes. Consistent with this, deletion of ant(9)-Ic in ZJ499 resulted in a specific and significant decrease in MIC of spectinomycin. Furthermore, the purified ANT(9)-Ic protein showed stringent substrate specificity for spectinomycin with a Km value of 44.83 μM and a kcat/Km of 2.8 × 104 M-1 s-1, echoing the above observations of susceptibility testing. In addition, comparative genomic analysis revealed that the genetic context of ant(9)-Ic was conserved in Brucella, with no mobile genetic elements found within its 20-kb surrounding region. Overall, our results demonstrate that ANT(9)-Ic is a novel member of the ANT(9)-I lineage, contributing to the intrinsic spectinomycin resistance of ZJ499. IMPORTANCE The emergence, evolution, and worldwide spread of antibiotic resistance present a significant global public health crisis. For aminoglycoside antibiotics, enzymatic drug modification is the most common mechanism of resistance. We identify a novel chromosomal aminoglycoside nucleotidyltransferase from B. intermedia, called ANT(9)-Ic, which shares the highest identity (47.44%) with the previously known ANT(9)-Ia and plays an important role in spectinomycin resistance of the host strain. Analysis of the genetic environment and origin of ant(9)-Ic shows that the gene and its surrounding region are widely conserved in Brucella, and no mobile elements are detected, indicating that ANT(9)-Ic may be broadly important in the natural resistance to spectinomycin of Brucella species.

Aprosamine Derivatives Active against Multidrug-Resistant Gram-Negative Bacteria

Novel aprosamine derivatives were synthesized for the development of aminoglycoside antibiotics active against multidrug-resistant Gram-negative bacteria. The synthesis of aprosamine derivatives involved glycosylation at the C-8' position and subsequent modification (epimerization and deoxygenation at the C-5 position and 1-N-acylation) of the 2-deoxystreptamine moiety. All 8'-β-glycosylated aprosamine derivatives (3a-h) showed excellent antibacterial activity against carbapenem-resistant Enterobacteriaceae and 16S ribosomal RNA methyltransferase-producing multidrug-resistant Gram-negative bacteria compared to the clinical drug, arbekacin. The antibacterial activity of 5-epi (6a-d) and 5-deoxy derivatives (8a,b and 8h) of β-glycosylated aprosamine was further enhanced. On the other hand, the derivatives (10a,b and 10h) in which the amino group at the C-1 position was acylated with (S)-4-amino-2-hydroxybutyric acid showed excellent activity (MICs 0.25-0.5 μg/mL) against resistant bacteria that produce the aminoglycoside-modifying enzyme, aminoglycoside 3-N-acetyltransferase IV, which induces high resistance against parent apramycin (MIC > 64 μg/mL). In particular, 8b and 8h showed approximately 2- to 8-fold antibacterial activity against carbapenem-resistant Enterobacteriaceae and 8- to 16-fold antibacterial activity against resistant Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, compared to apramycin. Our results showed that aprosamine derivatives have immense potential in the development of therapeutic agents for multidrug-resistant bacteria.

Molecular characterization of antibiotic resistance, aminoglycoside and PmrA genes among foodborne and clinical Acinetobacter spp.

The assessment of antibiotic resistance and related genes of foodborne Acinetobacter spp. and the analysis of whether they are genetically related to clinical infection-agent strains are crucial in terms of sustainability of food safety. The study at hand investigated antibiotic resistance, aminoglycoside-modifying enzyme (AME), and colistin resistance (PmrA) genes, clonal relationships while evaluating a possible correlation between antibiotic resistance and related genes between 27 foodborne and 50 clinical Acinetobacter spp. in Turkey. Antimicrobial susceptibilities, (AME), PmrA genes, and clonal relatedness of the strains were performed by disc diffusion, PCR, and Pulsed Field gel Electrophoresis (PFGE) methods, respectively. The aph-AI, aph-6, anth (3’’)-I, aadA1, aadB and PmrA genes were found as (24, 48%), (11, 22%), (7, 14%), (1, 2%), (2, 4%), and (46, 92%) respectively in clinical strains. This rate was found as (14, 51.9%), (16, 59.3%), (19, 70.4%), (2, 7.4%), (0, 0%), and (27, 100%) respectively in foodborne isolates. A positive correlation existed between the number of aph-AI gene positivity and trimethoprim-sulfamethoxazole and gentamycin resistance; anth (3’’)-I gene positivity, and colistin resistance; PmrA gene positivity and piperacillin-tazobactam, ceftazidime, meropenem, amikacin, and imipenem resistance in clinical strains (P

Clonal evolution and antimicrobial resistance of Acinetobacter baumannii isolates from Korean hospitals over the last decade.

The wide-spread of drug-resistant Acinetobacter baumannii is a global health problem. This study investigated the clonal distribution and antimicrobial resistance of 167 A. baumannii isolates from two Korean university hospitals from 2009 to 2019 by analyzing the sequence types (STs), antimicrobial resistance, and resistance determinants of carbapenems and aminoglycosides. Twenty STs, including 16 pre-existing STs and four unassigned STs, were identified in A. baumannii isolates using the Oxford multilocus sequence typing scheme. Two STs, ST191 (n=77) and ST451 (n=40), were prevalent, and majority (n=153) of the isolates belonged to clonal complex 208. The ST191 isolates were detected during the study period, whereas ST451 isolates were detected after 2016. One hundred forty-seven (87%) of 167 A. baumannii isolates were non-susceptible to carbapenems. The ST191 and ST451 isolates exhibited higher resistance to antimicrobial agents than that of the sporadic ST isolates. Interestingly, ST451 isolates exhibited lower susceptibility to minocycline and tigecycline than the other ST isolates. All carbapenem-non-susceptible A. baumannii isolates, except four, carried the ISAbaI-blaOXA-23 structure. armA was detected in all amikacin-non-susceptible isolates (n=128) except for one isolate. Five aminoglycoside-modifying enzyme (AME) genes were detected, but their carriage varied between STs; ant(3″)-Ia and aac(6')-Ib were more common in ST191 than in ST451, while aph(3')-Ia was more common in ST451 than in ST191. This study demonstrated the clonal evolution related to antimicrobial resistance in A. baumannii.