Aminoglycoside-modifying Enzymes Research Articles

Combating Aminoglycoside Resistance: From Structural and Functional Characterisation to Therapeutic Challenges with RKAAT.

A comprehensive knowledge of aminoglycoside-modifying enzymes (AMEs) and their role in bacterial resistance mechanisms is urgently required due to the rising incidence of antibiotic resistance, particularly in Klebsiella pneumoniae infections. This study explores the essential features of AMEs, including their structural and functional properties, the processes by which they contribute to antibiotic resistance, and the therapeutic importance of aminoglycosides. The study primarily examines the Recombinant Klebsiella pneumoniae Aminoglycoside Adenylyl Transferase (RKAAT), particularly emphasizing its biophysical characteristics and the sorts of resistance it imparts. Furthermore, this study examines the challenges presented by RKAAT-mediated resistance, an evaluation of treatment methods and constraints, and options for controlling infection. The analysis provides a prospective outlook on strategies to address and reduce antibiotic resistance. This extensive investigation seeks to provide vital insights into the continuing fight against bacterial resistance, directing future research efforts and medicinal approaches.

Prevalence of the CRISPR-cas system and its association with antibiotic resistance in clinical Klebsiella pneumoniae isolates

Background and objective(s)CRISPR-Cas is a prokaryotic adaptive immune system that protects bacteria and archaea against mobile genetic elements (MGEs) such as bacteriophages plasmids, and transposons. In this study, we aimed to assess the prevalence of the CRISPR-Cas systems and their association with antibiotic resistance in one of the most challenging bacterial pathogens, Klebsiella pneumoniae.Materials and methodsA total of 105 K. pneumoniae isolates were collected from various clinical infections. Extended-spectrum β-lactamases (ESBLs) phenotypically were detected and the presence of ESBL, aminoglycoside-modifying enzymes (AME), and CRISPR-Cas system subtype genes were identified using PCR. Moreover, the diversity of the isolates was determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR.ResultsPhenotypically, 41.9% (44/105) of the isolates were found to be ESBL producers. A significant inverse correlation existed between the subtype I-E CRISPR-Cas system’s presence and ESBL production in K. pneumoniae isolates. Additionally, the frequency of the ESBL genes blaCTX−M1 (3%), blaCTX−M9 (12.1%), blaSHV (51.5%), and blaTEM (33.3%), as well as some AME genes such as aac(3)-Iva (21.2%) and ant(2’’)-Ia (3%) was significantly lower in the isolates with the subtype I-E CRISPR-Cas system in comparison to CRISPR-negative isolates. There was a significant inverse correlation between the presence of ESBL and some AME genes with subtype I-E CRISPR-Cas system.ConclusionThe presence of the subtype I-E CRISPR-Cas system was correlated with the antibiotic-resistant gene (ARGs). The isolates with subtype I-E CRISPR-Cas system had a lower frequency of ESBL genes and some AME genes than CRISPR-negative isolates.

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). Aminogly- coside 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 investi- gated 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.

Design, Synthesis and Activity Evaluation Against Drug‐Resistant Bacteria of 6’‐Amidine Modified Aminoglycoside Derivatives

AbstractThe issue of bacterial resistance has become a matter of significant concern globally, prompting extensive research into the development and clinical application of aminoglycoside antibiotics (AGAs) as a crucial antibacterial therapy. One of the primary mechanisms contributing to aminoglycoside antibiotic resistance is the action of aminoglycoside modifying enzymes (AMEs), which modify aminoglycoside antibiotics and consequently confer drug resistance. Chemical modification of the AME action site can eliminate functional groups vulnerable to AMEs, thus improving drug resistance. This study involves the introduction of α‐hydroxy‐γ‐aminobutyric acid and amidine to the 1 and 6′ positions of sisomicin, respectively, to produce a range of 6′‐amidine modified sisomicin derivatives of AGAs. The research evaluates the antibacterial activity, absorption, distribution, metabolism, and excretion (ADME) properties, pharmacokinetic properties, cytotoxicity, nephrotoxicity, and drug resistance of AGAs. The outcomes of this study are significant in terms of enriching the structural understanding of aminoglycosides, providing insight into their structure–activity relationship, and producing new AGAs with high antibacterial activity, low ototoxicity, and nephrotoxicity.

Molecular Analysis of Carbapenem and Aminoglycoside Resistance Genes in Carbapenem-Resistant Pseudomonas aeruginosa Clinical Strains: A Challenge for Tertiary Care Hospitals.

Carbapenem-resistant Pseudomonas aeruginosa (P. aeruginosa) strains have become a global threat due to their remarkable capability to survive and disseminate successfully by the acquisition of resistance genes. As a result, the treatment strategies have been severely compromised. Due to the insufficient available data regarding P. aeruginosa resistance from Pakistan, we aimed to investigate the resistance mechanisms of 249 P. aeruginosa strains by antimicrobial susceptibility testing, polymerase chain reaction for the detection of carbapenemases, aminoglycoside resistance genes, extended-spectrum beta-lactamases (ESBLs), sequence typing and plasmid typing. Furthermore, we tested silver nanoparticles (AgNPs) to evaluate their in vitro sensitivity against antimicrobial-resistant P. aeruginosa strains. We observed higher resistance against antimicrobials in the general surgery ward, general medicine ward and wound samples. Phenotypic carbapenemase-producer strains comprised 80.7% (201/249) with 89.0% (179/201) demonstrating genes encoding carbapenemases: blaNDM-1 (32.96%), blaOXA48 (37.43%), blaIMP (7.26%), blaVIM (5.03%), blaKPC-2 (1.12%), blaNDM-1/blaOXA48 (13.97%), blaOXA-48/blaVIM (1.68%) and blaVIM/blaIMP (0.56%). Aminoglycoside-modifying enzyme genes and 16S rRNA methylase variants were detected in 43.8% (109/249) strains: aac(6')-lb (12.8%), aac(3)-lla (12.0%), rmtB (21.1%), rmtC (11.0%), armA (12.8%), rmtD (4.6%), rmtF (6.4%), rmtB/aac(3)-lla (8.2%), rmtB/aac(6')-lla (7.3%) and rmtB/armA (3.6%). In total, 43.0% (77/179) of the strains coharbored carbapenemases and aminoglycoside resistance genes with 83.1% resistant to at least 1 agent in 3 or more classes and 16.9% resistant to every class of antimicrobials tested. Thirteen sequence types (STs) were identified: ST235, ST277, ST234, ST170, ST381, ST175, ST1455, ST1963, ST313, ST207, ST664, ST357 and ST348. Plasmid replicon types IncFI, IncFII, IncA/C, IncL/M, IncN, IncX, IncR and IncFIIK and MOB types F11, F12, H121, P131 and P3 were detected. Meropenem/AgNPs and Amikacin/AgNPs showed enhanced antibacterial activity. We reported the coexistence of carbapenemases and aminoglycoside resistance genes among carbapenem-resistant P. aeruginosa with diverse clonal lineages from Pakistan. Furthermore, we highlighted AgNP's potential role in handling future antimicrobial resistance concerns.

First report of coexistence of blaKPC-2 and blaNDM-1 in carbapenem-resistant clinical isolates of Klebsiella aerogenes in Brazil.

Klebsiella aerogenes is an important opportunistic pathogen with the potential to develop resistance against last-line antibiotics, such as carbapenems, limiting the treatment options. Here, we investigated the antibiotic resistance profiles of 10 K. aerogenes strains isolated from patient samples in the intensive-care unit of a Brazilian tertiary hospital using conventional PCR and a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the blaKPC-2 and blaNDM-1 genes simultaneously. All isolates were completely resistant to β-lactam antibiotics, including ertapenem, imipenem, and meropenem with differencing levels of resistance to aminoglycosides, quinolones, and tigecycline also observed. Half of the strains studied were classified as multidrug-resistant. The carbapenemase-producing isolates carried many genes of interest including: β-lactams (blaNDM-1, blaKPC-2, blaTEM-1, blaCTX-M-1 group, blaOXA-1 group and blaSHVvariants in 20-80% of the strains), aminoglycoside resistance genes [aac(6')-Ib and aph(3')-VI, 70 and 80%], a fluoroquinolone resistance gene (qnrS, 80%), a sulfonamide resistance gene (sul-2, 80%) and a multidrug efflux system transporter (mdtK, 70%) while all strains carried the efflux pumps Acr (subunit A) and tolC. Moreover, we performed a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the blaKPC-2 and blaNDM-1 genes simultaneously. The draft genome assembly of the CRK317 had a total length of 5,462,831 bp and a GC content of 54.8%. The chromosome was found to contain many essential genes. In silico analysis identified many genes associated with resistance phenotypes, including β-lactamases (blaOXA-9, blaTEM-1, blaNDM-1, blaCTX-M-15, blaAmpC-1, blaAmpC-2), the bleomycin resistance gene (bleMBL), an erythromycin resistance methylase (ermC), aminoglycoside-modifying enzymes [aac(6')-Ib, aadA/ant(3")-Ia, aph(3')-VI], a sulfonamide resistance enzyme (sul-2), a chloramphenicol acetyltransferase (catA-like), a plasmid-mediated quinolone resistance protein (qnrS1), a glutathione transferase (fosA), PEtN transferases (eptA, eptB) and a glycosyltransferase (arnT). We also detected 22 genomic islands, eight families of insertion sequences, two putative integrative and conjugative elements with a type IV secretion system, and eight prophage regions. This suggests the significant involvement of these genetic structures in the dissemination of antibiotic resistance. The results of our study show that the emergence of carbapenemase-producing K. aerogenes, co-harboring blaKPC-2 and blaNDM-1, is a worrying phenomenon which highlights the importance of developing strategies to detect, prevent, and control the spread of these microorganisms.

Emergence of High-Level Antibiotic Resistance in Klebsiella pneumoniae: A Narrative Review

In an era marked by remarkable advancements in medicine, the persistent emergence of high-level antibiotic resistance in Klebsiella pneumoniae poses a critical threat to public health globally. As the worldwide spread of extensively drug-resistant (XDR) and pan-drug-resistant (PDR) K. pneumoniae strains continues to grow, a significant shift in how we approach treatment is on the horizon. The complex interaction of genetic factors, which encompasses a wide range of beta-lactamases, aminoglycoside-modifying enzymes, and chromosomal mutations, creates a dynamic resistance mechanism that counters the effects of antibiotics. These intricate adaptations, arising from both gene transfers facilitated by plasmids and changes in the genome itself, present a challenging obstacle to our efforts in managing antimicrobial effectiveness. Klebsiella infections come back stronger armed with molecular tactics that challenge healthcare systems, prolong hospital stays, and increase mortality. Beyond healthcare settings, the economic and social dimensions grow as resources are redirected, intensifying the impact on vulnerable groups. This review delves into the intricate mechanisms behind the high-level antibiotic resistance in K. pneumoniae, examining its epidemiological, molecular, and clinical facets. Highlighting the necessity for coordinated research, medical protocols, and policies, the review underscores the importance of judicious antibiotic utilization, drug innovation, and rigorous infection management.

Presence of genes encoding aminoglycoside-modifying enzyme (AME) and virulence factors in high-level aminoglycoside-resistant (HLAR) Enterococcus strains isolated from retail chicken meat in Turkey

In this study, the presence of aminoglycoside-modifying enzyme (AME) and virulence factor genes were investigated in previously isolated 32 high-level aminoglycoside-resistant (HLAR) Enterococcus strains isolated from retail chicken meat in Turkey. At least one AME-encoding gene was detected in HLAR enterococci by polymerase chain reaction (PCR). The ant(6ʹ)-Ia was identified as the most prevalent (87.5%, 28/32) AME gene. The aph(3ʹ)-IIIa (78.13%, 25/32), ant(4ʹ)-Ia (68.75%, 22/32), aph(2ʹʹ)-Ib (62.5%, 20/32), aac(6ʹ)-Ie-aph(2ʹʹ)-Ia (21.88%, 7/32) and aph(2ʹʹ)-Ic (9.38%, 3/32) are the other detected AME-encoding genes in strains. The aph(2ʹʹ)-Id was found in none of the HLAR strains. The aph(2ʹʹ)-Ib and ant(6ʹ)-Ia were identified as the most frequently AME-encoding genes in high-level gentamicin-resistant (HLGR) and high-level streptomycin-resistant (HLSR) strains, respectively. Among the 32 HLAR strains, only E. faecalis MSE61.1 and E. avium MSE63.1 were found capable of hydrolyzing gelatine. All HLAR strains showed α-hemolytic activity except E. durans MG13.4 and E. casseliflavus MGM111.1, which were exhibited β- and γ-hemolytic activity, respectively. It was determined that all HLAR strains, except E. durans MGE13.1 and MGE63.1, contain at least one virulence factor gene. The efaAfm (87.5%, 28/32), acm (65.63%, 21/32) and gelE (37.5%, 12/32) were found to be the most prevalent virulence factor genes. HLAR enterococci strains that have the virulence factor genes may pose a risk to consumer health.

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.