Aminoglycoside-modifying Enzyme Genes Research Articles

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.

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.

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.

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.

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

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].

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.

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.

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.

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.

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

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

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

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

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

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

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

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

Associated resistance of Escherichia coli isolated from humans and animals to polymyxin and beta-lactam antibiotics

Escherichia coli isolates from various sources from 2018 to 2019 were included in the study. Mcr-1 genes were found in two of 105 animal strains (2%) and seven of 928 human strains (0.8%). All mcr-1-positive strains showed a low level of resistance to colistin (MIC ranged from 4 to 8 µg/ml). Both strains isolated from animals remained sensitive to betalactam antibiotics and did not contain beta-lactamase genes. Beta-lactamases were absent only in one of the strains isolated from humans. Four strains were resistant to cephalosporins with sensitivity to carbapenems and carried class A (blaCTX-M-15 or blaCTX-M-1) or class C (blaCMY-2) extended-spectrum beta-lactamases genes. One strain showed resistance to cephalosporins and meropenem and contained four beta-lactamase genes: blaNDM-1, blaCTX-M-15, blaTEM-1B, and blaCMY-6. Only one strain isolated from animals remained sensitive to ciprofloxacin, the rest showed high level of resistance, had amino acid substitutions in the DNA gyrase genes or mutations leading to overexpression of the mdfA gene. In terms of resistance to aminoglycosides, the strains varied widely and carried up to four aminoglycoside-modifying enzyme genes. One strain isolated from humans showed resistance to tigecycline, but no genes conferring resistance to this antibiotic were found. The data obtained substantiate the need for extended studies on the molecular epidemiology of associated resistance to polymyxins and beta-lactams.

Whole Genome Sequence Analysis of Multidrug Resistant Escherichia coli and Klebsiella pneumoniae Strains in Kuwait.

The spread of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae is a global concern. The management of infections caused by multidrug resistance (MDR) isolates poses substantial clinical challenges in both hospitals and communities. This study aimed to investigate the genetic characteristics and variations of MDR E. coli and K. pneumoniae isolates. Bacterial identification and antibiotic susceptibility testing against 19 antibiotics were performed by standard methods. Whole genome sequencing (WGS) was carried out on eight carbapenem-resistant isolates using an Illumina MiSeq platform. The assembled draft genomes were annotated, then sequences were blasted against antimicrobial resistance (AMR) genes database. WGS detected several resistance genes mediating the production of β-lactamases, including carbapenems and extended-spectrum β-lactamase genes as (blaOXA-1/-48, blaKPC-2/-29, blaCMY-4/-6, blaSHV-11/-12, blaTEM-1, blaCTX-M-15, blaOKP-B, blaACT and blaEC). Furthermore quinolone resistance including oqxA/oqxB, aac(6′)-Ib-cr5, gyrA_D87N, gyrA_S83F, gyrA_S83L, parC_S80I, parE_S458A, parE_I355T, parC_S80I, and qnrB1. In addition to aminoglycoside modifying enzymes genes (aph(6)-Id, aph(3″)-Ib, aac(3)-IIa, aac(6′)-Ib, aadA1, aadA2 and aadA5), trimethoprim-sulfamethoxazole (dfrA12/A14/A17 and sul1/sul2), tetracycline (tetA and tetB), fosfomycin (fosA and uhpT_E350Q) resistance genes, while other genes were detected conferring chloramphenicol (floR, catA2, and efflux pump cmIA5), macrolides resistance (mph(A) and erm(B), and quaternary ammonium efflux pump qacEdelta. Bleomycin and colistin resistance genes were detected as ble and pmrB_R256G, respectively. Comprehensive analysis of MDR strains provided by WGS detected variable antimicrobial resistance genes and their precise resistance mechanism. WGS is essential for control and prevention strategies to combat the growing threat of AMR and the implementation of multifaceted interventions are needed.

Molecular Characterization of High-Level Aminoglycoside Resistance among Enterococcus Species.

Background Enterococci are nosocomial pathogen. They can develop high-level resistance to aminoglycoside by producing aminoglycoside modifying enzymes (AMEs). In enterococci, high level resistance to aminoglycosides is mediated by acquisition of plasmid mediated genes encoding for aminoglycoside modifying enzymes (AMEs). High level gentamicin resistance (MIC ≥ 500μg /mL) is predominantly mediated by aac(6′)-Ie-aph(2″)-Ia, encoding the bifunctional aminoglycoside modifying enzyme AAC(6′)-APH(2″). This enzyme eliminates the synergistic activity of gentamicin when combined with a cell wall active agent. Other AME genes such as aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id and ant(4′)-1a have also been detected in enterococci. Objective This study was carried out to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of Enterococci . Materials and Methods A total number of 150 clinical isolates were included in this study. Susceptibility to various antibiotics was determined by disc diffusion. Minimum Inhibitory Concentration (MIC) was ascertained by agar dilution method. Polymerase chain reaction was done to screen the following AMEs (aac(6′)-Ie-aph(2″)-Ia; aph(2″)-Ib; aph(2″)-Ic; aph(2″)-Id and aph(3′)- IIIa genes) . Results 51.3% of the study isolates exhibited high level gentamicin resistance. Polymerase chain reaction revealed that aph(3′)-111a is the most prevalent AME, followed by aac(6′)-1e-aph(2″)-1a . The combination of both the genes were detected in 44.1% of the study isolates. The rest of the AMEs and their combinations were not encountered in this study. 8.6% of the study isolates did not harbour any AME genes screened for, but was phenotypically resistant to gentamicin. In contrast 31.3% anchored the AME genes but phenotypically appeared susceptible to gentamicin. Conclusion This study indicates the high- level aminoglycoside resistance disseminated among Enterococci in our geographical region. It also emphasizes the detection of AMEs by PCR is mandatory because strains that appear susceptible by disc diffusion and/or MIC method may harbour one or more AMEs genes leading to therapeutic failure.

Evaluation of phenotypic and genotypic patterns of aminoglycoside resistance in the Gram-negative bacteria isolates collected from pediatric and general hospitals

The purpose of the current study was to evaluate the phenotypic and genotypic patterns of aminoglycoside resistance among the Gram-negative bacteria (GNB) isolates collected from pediatric and general hospitals in Iran. A total of 836 clinical isolates of GNB were collected from pediatric and general hospitals from January 2018 to the end of December 2019. The identification of bacterial isolates was performed by conventional biochemical tests. Susceptibility to aminoglycosides was evaluated by the disk diffusion method (DDM). The frequency of genes encoding aminoglycoside-modifying enzymes (AMEs) was screened by the PCR method via specific primers. Among all pediatric and general hospitals, the predominant GNB isolates were Acinetobacter spp. (n = 327) and Escherichia coli (n = 144). However, E. coli (n = 20/144; 13.9%) had the highest frequency in clinical samples collected from pediatrics. The DDM results showed that 64.3% of all GNB were resistant to all of the tested aminoglycoside agents. Acinetobacter spp. and Klebsiella pneumoniae with 93.6%, Pseudomonas aeruginosa with 93.4%, and Enterobacter spp. with 86.5% exhibited very high levels of resistance to gentamicin. Amikacin was the most effective antibiotic against E. coli isolates. In total, the results showed that the aac (6')-Ib gene with 59% had the highest frequency among genes encoding AMEs in GNB. The frequency of the surveyed aminoglycoside-modifying enzyme genes among all GNB was found as follows: aph (3')-VIe (48.7%), aadA15 (38.6%), aph (3')-Ia (31.3%), aph (3')-II (14.4%), and aph (6) (2.6%). The obtained data demonstrated that the phenotypic and genotypic aminoglycoside resistance among GNB was quite high and it is possible that the resistance genes may frequently spread among clinical isolates of GNB.