Aminoglycoside Phosphotransferase Research Articles

Epidemiology of antimicrobial resistance in commensal E. coli from healthy dairy cattle on a Mediterranean pasture-based system of Australia: A cross-sectional study

This study aimed to determine the prevalence of antimicrobial resistance (AMR) in commensal E. coli from healthy lactating cows and calves in the Mediterranean pasture-based feeding dairy system of Western Australia (WA). Fecal samples were collected from healthy adult lactating cows and healthy calves from dairy farms in WA. Presumptive commensal E. coli was isolated from these samples and confirmed using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Broth microdilution was used to assess the prevalence and the phenotypic AMR profiles of the E. coli isolates to 8 antimicrobial agents of dairy industry and human importance. The minimum inhibitory concentration (MIC) for each isolate was interpreted using the Epidemiologic Cutoff (ECOFF) and Clinical and Laboratory Standards Institute (CLSI) breakpoints. Genomic characterization provided multi-locus sequence types and AMR genes for a selection of isolates categorised as non-wild type (NWT) by ECOFF values for the combination of ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. From a total of 1,117 fecal samples (633 adult, 484 calf) collected across 26 randomly selected farms, 891 commensal E. coli isolates were recovered (541 adult, 350 calf). Commensal E. coli classified as NWT was highest for ampicillin for both adult (68.8%; 95% CI = 64.7 - 72.7) and calf feces (67.1%; 95% CI = 62.0 - 72.0). A large proportion of tetracycline NWT and trimethoprim-sulfamethoxazole NWT organisms were also identified from calf feces, being 44.0% (95% CI = 38.7 - 49.4) and 24.6% (95% CI = 20.2 - 29.4) respectively. Clinical resistance prevalence was low, being higher for calves than for adult feces (ampicillin (adult: 7.8% (95% CI = 5.7 - 10.3); calf: 30.0% (95% CI = 25.2 - 35.1), tetracycline (adult: 6.3% (95% CI = 4.4 - 8.7); calf: 40.3% (95% CI = 35.1 - 45.6), and trimethoprim-sulfamethoxazole (adult: 2.6% (95% CI = 1.4 - 4.3); calf: 22.0% (95% CI = 17.7 - 26.7)). Commensal E. coli originating from calf feces was significantly higher in NWT prevalence compared with adult feces for ciprofloxacin (P = 0.023), gentamicin (P = 0.02), tetracycline (P < 0.001), and trimethoprim-sulfamethoxazole (P < 0.001). The overall number of antimicrobials an isolate was classified as NWT toward varied among farms and was significantly higher for isolates originating from calf than adult feces (P < 0.001). The strain type and sampling source of the commensal E. coli investigated were both associated with the commonality of the resultant resistance genome. Clinical resistance and NWT classification were highest for ampicillin, tetracycline, and trimethoprim-sulfamethoxazole, all antimicrobials commonly used in the treatment of dairy cattle in Australia. Though highly variable across farms, commensal E. coli isolated from healthy dairy calf feces had significantly higher NWT and multidrug resistance (MDR) prevalence compared with feces from healthy adult lactating dairy cows. The resistant genome identified in MDR isolates, though not always consistent with the phenotype, included QnrS1 and genes encoding AmpC β-lactamase and aminoglycoside phosphotransferase.

Spectral, XRD, morphological, DFT, and molecular modeling investigations of newly prepared isatin Schiff base complexes

The new Schiff base ligand was designed from a condensation reaction of isatin with 5‐amino‐2‐hydroxy benzoic acid. Then, the metal complexes were prepared. The structures of the prepared compounds were framed from thermogravimetric analyses (TG–DTG), mass spectrometry, elemental analyses, 1H‐NMR, IR, electronic spectra, UV–vis, and conductivity measurements. Surface and size morphology have been studied by X‐ray powder diffraction and scanning electron microscope (SEM) analysis. The IR spectra suggested that the ligand acted as bidentate coordinated with metal ions through deprotonated carboxylate oxygen atom and oxygen of phenolic group. With the exception of the complexes of Cr (III), Fe (III), and Cd (II), which were non‐electrolytes, the molar conductivities demonstrated the electrolytic behavior of all the rest complexes. The X‐ray diffraction (XRD) analysis patterns for the Schiff base complexes indicated their crystalline nature except the Co (II) complex which had amorphous structure. The ligand and its metal complexes were screened for their antimicrobial activities against two G‐positive, two G‐negative bacteria and against Aspergillus fumigatus and Candida albicans fungi. Additionally, the compounds were evaluated against the MFC‐7 breast cancer cell line for their anticancer activity. The outcomes demonstrated their strong antitumor activity. [Fe(HL)Cl2(H2O)2]0.5H2O complex has the lowest IC50 value = 9 μg mL−1. Molecular modeling studies for the ligand and its Co (II) and Fe (III) complexes were accomplished from density functional theory (DFT) analysis. From molecular docking study, the electrophilic moieties in both complexes displayed good binding interactions with the CDK6, aminoglycoside phosphotransferase, and CYP51 targeted proteins.

Investigating antimicrobial resistance genes in Kenya, Uganda and Tanzania cattle using metagenomics.

Antimicrobial resistance (AMR) is a growing problem in African cattle production systems, posing a threat to human and animal health and the associated economic value chain. However, there is a poor understanding of the resistomes in small-holder cattle breeds in East African countries. This study aims to examine the distribution of antimicrobial resistance genes (ARGs) in Kenya, Tanzania, and Uganda cattle using a metagenomics approach. We used the SqueezeMeta-Abricate (assembly-based) pipeline to detect ARGs and benchmarked this approach using the Centifuge-AMRplusplus (read-based) pipeline to evaluate its efficiency. Our findings reveal a significant number of ARGs of critical medical and economic importance in all three countries, including resistance to drugs of last resort such as carbapenems, suggesting the presence of highly virulent and antibiotic-resistant bacterial pathogens (ESKAPE) circulating in East Africa. Shared ARGs such as aph(6)-id (aminoglycoside phosphotransferase), tet (tetracycline resistance gene), sul2 (sulfonamide resistance gene) and cfxA_gen (betalactamase gene) were detected. Assembly-based methods revealed fewer ARGs compared to read-based methods, indicating the sensitivity and specificity of read-based methods in resistome characterization. Our findings call for further surveillance to estimate the intensity of the antibiotic resistance problem and wider resistome classification. Effective management of livestock and antibiotic consumption is crucial in minimizing antimicrobial resistance and maximizing productivity, making these findings relevant to stakeholders, agriculturists, and veterinarians in East Africa and Africa at large.

The Peptide Antibiotic Corramycin Adopts a β-Hairpin-like Structure and Is Inactivated by the Kinase ComG.

The rapid development of antibiotic resistance, especially among difficult-to-treat Gram-negative bacteria, is recognized as a serious and urgent threat to public health. The detection and characterization of novel resistance mechanisms are essential to better predict the spread and evolution of antibiotic resistance. Corramycin is a novel and modified peptidic antibiotic with activity against several Gram-negative pathogens. We demonstrate that the kinase ComG, part of the corramycin biosynthetic gene cluster, phosphorylates and thereby inactivates corramycin, leading to the resistance of the host. Remarkably, we found that the closest structural homologues of ComG are aminoglycoside phosphotransferases; however, ComG shows no activity toward this class of antibiotics. The crystal structure of ComG in complex with corramycin reveals that corramycin adopts a β-hairpin-like structure and allowed us to define the changes leading to a switch in substrate from sugar to peptide. Bioinformatic analyses suggest a limited occurrence of ComG-like proteins, which along with the absence of cross-resistance to clinically used drugs positions corramycin as an attractive antibiotic for further development.

Plasmid-free cheater cells commonly evolve during laboratory growth.

It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, extracellular beta-lactamases produced by resistant cells that subsequently degrade penicillin and related antibiotics allow neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show in multiple bacterial species that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface-grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss was still observed. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated.IMPORTANCEPlasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid-containing cell is grown in the presence of antibiotic. Here, we find that growth of plasmid-containing bacteria on a surface and to a lesser extent in liquid culture in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation.

A UPLC-based activity assay method for aminoglycoside phosphotransferase 4-Ia (APH4): A Selectable marker of plant transformation

The aminoglycoside phosphotransferase 4-Ia (APH4) is a hygromycin B phosphotransferase and catalyzes the phosphorylation of the 4-hydroxyl group of the antibiotic hygromycin B, inactivating its antibiotic activity. Therefore, APH4 has utility as a selectable marker for transformation of many plant species. However, suitable methods to measure and quantify plants expressing APH4 enzymes are lacking because of technical challenges, associated with both specificity and sensitivity. Here we describe a highly specific and sensitive APH4 enzymatic activity assay by measuring the production of adenosine 5′-diphosphate (ADP) using ultra performance liquid chromatography (UPLC)-based method. The present assay enabled determination of enzymatic activity of APH4 over a concentration range from 0.05 to 0.8 μg APH4/ml. Method performance validation sample were examined at five concentrations in triplicate in six independent sessions. Average CVs of 5.1 % for intra-assay and 11.7 % for inter-assay over all samples were determined for precision, and 6.0 % was determined for accuracy. Additionally, the method was validated for use to determine APH4 enzymatic activity in both microbially produced and plant-derived samples.

Synthesis and Evaluation of Novel 5‐O‐(4‐C‐Aminoalkyl‐β‐D‐ribofuranosyl) Apramycin Derivatives for the Inhibition of Gram‐Negative Pathogens Carrying the Aminoglycoside Phosphotransferase(3′)‐Ia Resistance Determinant

AbstractWe report the synthesis and evaluation of two new apramycin 5‐O‐β‐d‐ribofuranosides, or apralogs, carrying aminoalkyl branches at the ribofuranose 4‐position. This novel modification conveys excellent activity for the inhibition of protein synthesis by wild‐type bacterial ribosomes and correspondingly high antibacterial activity against several Gram‐negative pathogens. Notably, these new modifications overcome the reduction of antibacterial activity in other 2‐deoxystreptamine‐type aminoglycosides carrying a 5‐O‐ribofuranosyl moiety when challenged by the presence of an aminoglycoside phosphotransferase enzyme capable of acting on the ribose 5‐position.

Gut microbiota signature in children with autism spectrum disorder who suffered from chronic gastrointestinal symptoms

BackgroundChildren diagnosed with autism spectrum disorder (ASD) frequently suffer from persistent gastrointestinal symptoms, such as constipation and diarrhea. Various studies have highlighted differences in gut microbiota composition between individuals with ASD and healthy controls of similar ages. However, it’s essential to recognize that these disparities may be influenced by cultural practices, dietary habits, and environmental factors.MethodsIn this study, we collected fecal samples from both children diagnosed with ASD (n = 42) and healthy individuals (n = 41) residing in the southeastern coastal region of China. Subsequently, 16 S rRNA gene sequencing and advanced bioinformatics analyses were conducted to investigate the distinctive features of gut microbial communities within each group.ResultsThe ASD group consisted of 28 males and 14 females, with a median age of 5.8 years, while the control group included 25 males and 16 females, with a median age of 6.8 years. Among the 83 sequenced fecal samples, a total of 1031 operational taxonomic units (OTUs) were identified. These included 122 unique OTUs specific to the control group and 285 unique OTUs specific to the ASD group. Analyses of α-diversity and β-diversity unveiled significant differences in the abundance and composition of gut microbiota between the two groups. It was found that the dominant bacterial taxa in healthy individuals were UBA1819, Flavonifractor, and Bradyrhizobium. In contrast, the ASD group exhibited a prevalence of Streptococcus, Ruminococcus, and Ruminiclostridium. Further analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups (COG) showed significant differences in the metabolic functionalities of the gut microbiota between the two groups. Notably, the metabolic pathway related to alpha-linolenic acid (ALA) in the gut microbiota of the ASD group was notably diminished compared to the control group. Conversely, the ASD group demonstrated significantly elevated levels of metabolic pathways involving uncharacterized conserved proteins, aminoglycoside phosphotransferase, and inorganic pyrophosphatase compared to the control group.ConclusionsOverall, these results confirm that there are significant differences in the gut microbiota structure between children with ASD and healthy controls in the southeast coastal region of China. This underscores the critical significance of delving into clinical interventions capable of mitigating the gastrointestinal and psychological symptoms encountered by children with ASD. A particularly encouraging path for such interventions lies in the realm of fecal microbiota transplantation, a prospect that merits deeper inquiry.

Alpha-Naphthoflavone as a Novel Scaffold for the Design of Potential Inhibitors of the APH(3')-IIIa Nucleotide-Binding Site of Enterococcus faecalis.

The spread of nosocomial infections caused by antibiotic-resistant Enterococcus faecalis is one of the major threats to global health at present. While aminoglycosides are often used to combat these infections, their effectiveness is reduced by various resistance mechanisms, including aminoglycoside modifying enzymes, and there are currently no drugs to inhibit these enzymes. To address this issue, this study was conducted to identify potential aminoglycoside adjuvants from a database of 462 flavones. The affinity of these molecules with the nucleotide-binding site (NBS) of aminoglycoside phosphotransferase type IIIa of E. faecalis (EfAPH(3')-IIIa) was evaluated, and the five molecules with the highest binding energies were identified. Of these, four were naphthoflavones, suggesting that their backbone could be useful in designing potential inhibitors. The highest-ranked naphthoflavone, 2-phenyl-4H-benzo[h]chromen-4-one, was modified to generate two new derivatives (ANF2OHC and ANF2OHCC) to interact with the NBS similarly to adenine in ATP. These derivatives showed higher binding free energies, better stability in molecular dynamics analysis and superior pharmacokinetic and toxicological profiles compared to the parent molecule. These findings suggest that these alpha-naphthoflavone derivatives are potential inhibitors of EfAPH(3')-IIIa and that this core may be a promising scaffold for developing adjuvants that restore the sensitivity of aminoglycosides.

Three genes controlling streptomycin susceptibility in Agrobacterium fabrum.

Streptomycin (Sm) is a commonly used antibiotic for its efficacy against diverse bacteria. The plant pathogen Agrobacterium fabrum is a model for studying pathogenesis and interkingdom gene transfer. Streptomycin-resistant variants of A. fabrum are commonly employed in genetic analyses, yet mechanisms of resistance and susceptibility to streptomycin in this organism have not previously been investigated. We observe that resistance to a high concentration of streptomycin arises at high frequency in A. fabrum, and we attribute this trait to the presence of a chromosomal gene (strB) encoding a putative aminoglycoside phosphotransferase. We show how strB, along with rpsL (encoding ribosomal protein S12) and rsmG (encoding a 16S rRNA methyltransferase), modulates streptomycin sensitivity in A. fabrum. IMPORTANCE The plant pathogen Agrobacterium fabrum is a widely used model bacterium for studying biofilms, bacterial motility, pathogenesis, and gene transfer from bacteria to plants. Streptomycin (Sm) is an aminoglycoside antibiotic known for its broad efficacy against gram-negative bacteria. A. fabrum exhibits endogenous resistance to somewhat high levels of streptomycin, but the mechanism underlying this resistance has not been elucidated. Here, we demonstrate that this resistance is caused by a chromosomally encoded streptomycin-inactivating enzyme, StrB, that has not been previously characterized in A. fabrum. Furthermore, we show how the genes rsmG, rpsL, and strB jointly modulate streptomycin susceptibility in A. fabrum.

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.

The genome of Symbiodiniaceae-associated Stutzerimonas frequens CAM01 reveals a broad spectrum of antibiotic resistance genes indicating anthropogenic drift in the Palk Bay coral reef of south-eastern India.

An increase in antibiotic pollution in reef areas will lead to the emergence of antibiotic-resistant bacteria, leading to ecological disturbances in the sensitive coral holobiont. This study provides insights into the genome of antibiotics-resistant Stutzerimonas frequens CAM01, isolated from Favites-associated Symbiodiniaceae of a near-shore polluted reef of Palk Bay, India. The draft genome contains 4.67 Mbp in size with 52 contigs. Further genome analysis revealed the presence of four antibiotic-resistant genes, namely, adeF, rsmA, APH (3")-Ib, and APH (6)-Id that provide resistance by encoding resistance-nodulation-cell division (RND) antibiotic efflux pump and aminoglycoside phosphotransferase. The isolate showed resistance against 73% of the antibiotics tested, concurrent with the predicted AMR genes. Four secondary metabolites, namely Aryl polyene, NRPS-independent-siderophore, terpenes, and ectoine were detected in the isolate, which may play a role in virulence and pathogenicity adaptation in microbes. This study provides key insights into the genome of Stutzerimonas frequens CAM01 and highlights the emergence of antibiotic-resistant bacteria in coral reef ecosystems.

Resistom &lt;i&gt;Streptomyces rimosus&lt;/i&gt; - a reservoir of resistance genes to aminoglycoside antibiotics

The study of aminoglycoside acetyltransferases in actinobacteria of the genus Streptomyces is an integral part of the study of soil bacteria as the main reservoir and possible source of drug resistance genes. Previously, in the strain Streptomyces rimosus ATCC 10970 (producing oxyteteracycline), which is resistant to most natural aminoglycoside antibiotics, we have identified and biochemically characterized 3 aminoglycoside phosphotransferases, which cause resistance to kanamycin, neomycin, paromomycin, streptomycin, and hygromycin B. In the presented work, it was shown that resistance to other AGs in this strain is associated with the presence of the enzyme aminoglycoside acetyltransferase, belonging to the AAC(2′) subfamily. Induction of the expression of the gene, designated by us as aac(2′)-If, in Escherichia coli cells determines resistance to a wide range of natural aminoglycoside antibiotics (neomycin, gentamicin, tobramycin, sisomycin, and paromomycin) and to an increase in the minimum inhibitory concentrations of these antibiotics.

Resistome in Streptomyces rimosus - A Reservoir of Aminoglycoside Antibiotics Resistance Genes.

Investigation of aminoglycoside acetyltransferases in actinobacteria of the genus Streptomyces is an integral part of the study of soil bacteria as the main reservoir and possible source of drug resistance genes. Previously, we have identified and biochemically characterized three aminoglycoside phosphotransferases, which cause resistance to kanamycin, neomycin, paromomycin, streptomycin, and hygromycin B in the strain Streptomyces rimosus ATCC 10970 (producing oxytetracycline), which is resistant to most natural aminoglycoside antibiotics. In the presented work, it was shown that the resistance of this strain to other AGs is associated with the presence of the enzyme aminoglycoside acetyltransferase, belonging to the AAC(2') subfamily. Induction of the expression of the gene, designated by us as aac(2')-If, in Escherichia coli cells determines resistance to a wide range of natural aminoglycoside antibiotics (neomycin, gentamicin, tobramycin, sisomycin, and paromomycin) and increases minimum inhibitory concentrations of these antibiotics.

Functional characterization of a novel aminoglycoside phosphotransferase, APH(9)-Ic, and its variant from Stenotrophomonas maltophilia.

The intrinsic resistance mechanism plays an essential role in the bacterial resistance to a variety of the antimicrobials. The aim of this study is to find the chromosome-encoded novel antimicrobial resistance gene in the clinical isolate. The function of the predicted resistance gene was verified by gene cloning and antibiotic susceptibility test. Recombinant protein expression and enzyme kinetic studies were performed to explore the in vivo activity of the enzyme. Expression of the resistance gene exposed to antimicrobial was determined by RT-qPCR. Whole genome sequencing and bioinformatic analysis were applied to analyze the genetic context of the resistance gene. The novel aminoglycoside (AG) resistance genes designated aph(9)-Ic and aph(9)-Ic1 confer resistance to spectinomycin, and a recombinant strain harboring aph(9)-Ic (pMD19-T-aph(9)-Ic/DH5α) showed a significantly increased minimum inhibitory concentration (MIC) level against spectinomycin compared with the control strains (DH5α and pMD19-T/DH5α). The result of the kinetic analysis of APH(9)-Ic was consistent with the MIC result for the recombinant pMD19-T-aph(9)-Ic/DH5α, showing the efficient catalytic activity for spectinomycin [kcat/Km ratio = (5.58 ± 0.31) × 104 M-1·s-1]. Whole-genome sequencing demonstrated that the aph(9)-Ic gene was located on the chromosome with a relatively conserved genetic environment, and no mobile genetic element was found in its surrounding region. Among all the function-characterized resistance genes, APH(9)-Ic shares the highest amino acid sequence identity of 33.75% with APH(9)-Ia. We characterized a novel AG resistance gene aph(9)-Ic and its variant aph(9)-Ic1 that mediated spectinomycin resistance from S. maltophilia. The identification of the novel AG resistance genes will assist us in elucidating the complexity of resistance mechanisms in microbial populations.

Structure elucidation study of aminoglycoside phosphotransferase from B. cereus sensu lato: a comprehensive outlook for drug discovery

Structure elucidation study of aminoglycoside phosphotransferase from B. cereus sensu lato: a comprehensive outlook for drug discovery

Arctic Psychrotolerant Pseudomonas sp. B14-6 Exhibits Temperature-Dependent Susceptibility to Aminoglycosides.

Bacteria can evade antibiotics by acquiring resistance genes, as well as switching to a non-growing dormant state without accompanying genetic modification. Bacteria in this quiescent state are called persisters, and this non-inheritable ability to withstand multiple antibiotics is referred to as antibiotic tolerance. Although all bacteria are considered to be able to form antibiotic-tolerant persisters, the antibiotic tolerance of extremophilic bacteria is poorly understood. Previously, we identified the psychrotolerant bacterium Pseudomonas sp. B14-6 from the glacier foreland of Midtre Lovénbreen in High Arctic Svalbard. Herein, we investigated the resistance and tolerance of Pseudomonas sp. B14-6 against aminoglycosides at various temperatures. This bacterium was resistant to streptomycin and susceptible to apramycin, gentamicin, kanamycin, and tobramycin. The two putative aminoglycoside phosphotransferase genes aph1 and aph2 were the most likely contributors to streptomycin resistance. Notably, unlike the mesophilic Pseudomonas aeruginosa PA14, this cold-adapted bacterium demonstrated reduced susceptibility to all tested aminoglycosides in a temperature-dependent manner. Pseudomonas sp. B14-6 at a lower temperature formed the persister cells that shows tolerance to the 100-fold minimum inhibitory concentration (MIC) of gentamicin, as well as the partially tolerant cells that withstand 25-fold MIC gentamicin. The temperature-dependent gentamicin tolerance appears to result from reduced metabolic activity. Lastly, the partially tolerant Pseudomonas sp. B14-6 cells could slowly proliferate under the bactericidal concentrations of aminoglycosides. Our results demonstrate that Pseudomonas sp. B14-6 has a characteristic ability to form cells with a range of tolerance, which appears to be inversely proportional to its growth rate.

Dual-Mechanism Confers Self-Resistance to the Antituberculosis Antibiotic Capreomycin.

Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from Saccharothrix mutabilis subspecies capreolus. The gene cluster for CMN biosynthesis has been identified and sequenced, wherein the cph gene was annotated as a phosphotransferase likely engaging in self-resistance. Previous studies reported that Cph inactivates two CMNs, CMN IA and IIA, by phosphorylation. We, herein, report that (1) Escherichia coli harboring the cph gene becomes resistant to both CMN IIA and IIB, (2) phylogenetic analysis regroups Cph to a new clade in the phosphotransferase protein family, (3) Cph shares a three-dimensional structure akin to the aminoglycoside phosphotransferases with a high binding affinity (KD) to both CMN IIA and IIB at micromolar levels, and (4) Cph utilizes either ATP or GTP as a phosphate group donor transferring its γ-phosphate to the hydroxyl group of CMN IIA. Until now, Cph and Vph (viomycin phosphotransferase) are the only two known enzymes inactivating peptide-based antibiotics through phosphorylation. Our biochemical characterization and structural determination conclude that Cph confers the gene-carrying species resistance to CMN by means of either chemical modification or physical sequestration, a naturally manifested belt and braces strategy. These findings add a new chapter into the self-resistance of bioactive natural products, which is often overlooked while designing new bioactive molecules.

Fate of bacterial community, antibiotic resistance genes and gentamicin residues in soil after three-year amendment using gentamicin fermentation waste

Over a three-year field trial, the impacts of composted and raw gentamicin fermentation waste (GFW) application to land on residual soil gentamicin levels, physicochemical properties, bacterial community composition, and antibiotic resistance genes (ARGs) were assessed. In the saline-alkali soil tested, GFW application decreased electrical conductivity (EC) and pH. Importantly, there was no measurable long-term accumulation of gentamicin as a result of GFW addition. Changes in the abundance of Bacillus was primarily associated with degradation of gentamicin in soil, whereas wider (i.e. more general) shifts in bacterial communities over the treatments was linked to alteration of soil physicochemical properties, particularly pH, total nitrogen, dissolved organic carbon, EC, NO3−-N and NH4+-N. Compared with other treatments, soils receiving composted GFW harbored more types of ARGs and significantly higher (P < 0.05) abundances of mobile genes elements (MGEs) (especially IncQ and Int1) and aminoglycoside ARGs (especially aminoglycoside phosphotransferases genes, APH). Finally, the abundances of ARGs in soils receiving raw and composted GFW were 59.60% and 50.26% higher than that in soils only receiving chemical fertilizer, respectively. Specifically, the abundances of APH, especially strB, were significantly higher than other kinds of ARGs (P < 0.05). The results of linear regression and partial least squares path model showed that MGEs, including plasmids, integrons, and transposons, along with soil properties (EC and NH4+-N) were the main factors associated with change in ARGs. Furthermore, different MGEs were involved in different transfer mechanisms of specific ARGs. Our findings demonstrated the potential risks of using raw and composted GFW as fertilizer, and suggest potential solutions to this problem.

Identification and characteristics of a novel aminoglycoside phosphotransferase, APH(3')-IId, from an MDR clinical isolate of Brucella intermedia.

To describe a novel chromosomal aminoglycoside phosphotransferase named APH(3')-IId identified in an MDR Brucella intermedia ZJ499 isolate from a cancer patient. Species identity was determined by PCR and MALDI-TOF MS analysis. WGS was performed to determine the genetic elements conferring antimicrobial resistance. Gene cloning, transcriptional analysis and targeted gene deletion, as well as protein purification and kinetic analysis, were performed to investigate the mechanism of resistance. APH(3')-IId consists of 266 amino acids and shares the highest identity (48.25%) with the previously known APH(3')-IIb. Expression of aph(3')-IId in Escherichia coli decreased susceptibility to kanamycin, neomycin, paromomycin and ribostamycin. The aph(3')-IId gene in ZJ499 was transcriptionally active under laboratory conditions and the relative abundance of this transcript was unaffected by treatment with the above four antibiotics. However, deletion of aph(3')-IId in ZJ499 results in decreased MICs of these drugs. The purified APH(3')-IId showed phosphotransferase activity against kanamycin, neomycin, paromomycin and ribostamycin, with catalytic efficiencies (kcat/Km) ranging from ∼105 to 107 M-1 s-1. Genetic environment and comparative genomic analyses suggested that aph(3')-IId is probably a ubiquitous gene in Brucella, with no mobile genetic elements detected in its surrounding region. APH(3')-IId is a novel chromosomal aminoglycoside phosphotransferase and plays an important role in the resistance of B. intermedia ZJ499 to kanamycin, neomycin, paromomycin and ribostamycin. To the best of our knowledge, APH(3')-IId represents the fourth characterized example of an APH(3')-II enzyme.