Aminoglycoside-modifying Enzyme Genes Research Articles

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.

Resistance traits and molecular characterization of multidrug-resistant Acinetobacter baumannii isolates from an intensive care unit of a tertiary hospital in Guangdong, southern China.

This study aims to characterize antimicrobial resistance (AMR) of all the non-duplicated Acinetobacter baumannii strains isolated from an intensive care unit in a tertiary hospital during the period of January 1 to December 31, 2015. A. baumannii (n = 95 strains) isolated from patients was subjected to antimicrobial susceptibility test (AST) by Vitek 2 Compact system to determine minimum inhibitory concentrations, followed by genotyping by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). Resistance genes of interest were PCR amplified and sequenced. All isolates were qualified as MDR, with a resistance rate of > 80% to 8 antimicrobials tested. In terms of beta-lactamase detection, the blaOXA23, blaTEM-1, and armA genes were detected frequently at 92.63%, 9 1.58%, and 88.42%, respectively. The metallo-β-lactamase genes blaIMP and blaVIM were undetected. Aph (3')-I was detected in 82 isolates (86.32%), making it the most prevalent aminoglycoside-modifying enzyme (AMEs) encoding gene. In addition, ant (3″)-I was detected at 30.53%, while 26.32% of the strains harbored an aac (6')-Ib gene. ERIC-PCR typing suggested moderate genetic diversity among the isolates, which might be organized into 10 distinct clusters, with cluster A (n = 86 isolates or 90.53%) being the dominant cluster. All of the A. baumannii strains detected in the ICU were MDR clones exhibiting extremely high resistance to carbapenems and aminoglycosides as monitored throughout the study period. They principally belonged to a single cluster of isolates carrying blaOXA23 and armA co-producing different AMEs genes.

In Vitro Activity of Plazomicin Against Carbapenem Resistant Klebsiella pneumoniae Strains.

In our study, we evaluated the in vitro activities of plazomicin, amikacin, gentamicin, and tobramycin among fifty carbapenem resistant Klebsiella pneumoniae (CR-Kp) isolates. Aminoglycoside resistance genes in selected CR-Kp strains were also examined. Minimum inhibitory concentration (MIC) of meropenem, plazomicin, tobramycin, gentamicin, and amikacin were determined by gradient test (G-test) method. In all strains carbapenemase activity was assessed by polymerase chain reaction (PCR). Aminoglycoside modifying enzyme (AME) genes in 14 CR-Kp strains that are resistant to at least one of tobramycin, gentamicin, and amikacin among fifty CR-Kp isolates, and 16S ribosomal methylase genes in 6 CR-Kp strains with plazomicin MIC ≥ 128 mg/L were investigated by PCR method. The most frequently detected carbapenemase enzyme in the strains in our study was OXA-48 (88%). Aminoglycoside susceptibilities of all isolates were determined; plazomicin 84%, amikacin 66%, gentamicin 50%, tobramycin 18%. The most common AME gene positivities were found, 93% (n = 13) ant(3')-I, 78% (n = 11) aac(6')-Ib, 57% (n = 8) aac(3')-IV, 42% (n = 6) aac(3')-IIa, and 29% (n = 4) aph(3')-VI. Most of the isolates examined for the presence of AME carry at least two or more AME genes. The most common 16S ribosomal methylase gene was rmtH. In our study, MIC values of ≥ 256 µg/mL were found in 6 (12%) of 50 isolates against amikacin, tobramycin, and gentamicin, including plazomicin. At least two 16S ribosomal methylase gene positivity has been shown in these 6 strains. In our study, increased in vitro efficacy of plazomicin was shown in CR-Kp isolates comparing to other aminoglycosides. Plazomicin is an effective treatment option against CR-Kp isolates and needs to be sup-ported by clinical studies.

Antibiotic Resistance Determinants and Virulence Factors of Hypervirulent and Carbapenem Non-Susceptible Pseudomonas aeruginosa

The aim was to investigate the distribution of antibiotic resistance determinants and virulence factors in a group of carbapenem non-susceptible Pseudomonas aeruginosa (P. aeruginosa). From March 2018 to May 2019, a total of 98 P. aeruginosa samples were collected from 6 hospitals in Ningbo and Hangzhou, Zhejiang Province, China. Drug susceptibility tests to 13 antimicrobial agents were conducted. The presence of antibiotic resistance determinants and virulence factors were investigated by PCR, including 39 β-lactamase genes, 14 aminoglycoside modifying enzyme genes, 10 16SrRNA methylase genes, and 11 virulence genes. Phylogenetics of 98 P. aeruginosa was analyzed by sample cluster analysis (UPGMA). PCR revealed the presence of 7 β-lactamase genes, 5 aminoglycoside modifying enzymes, 1 16S rRNA methylase gene, and 8 virulence genes in total, at least 2 β-lactamase genes and 4 virulence genes were positive in every isolate. In addition, regional differences in distributions of resistance and virulence genes remained between 2 cities. Sample cluster analysis showed that the strains had obvious aggregation and were divided into several clusters, strains in the same cluster were isolated from different hospitals, even from different cities. Carrying resistance genes blaPDC and blaOXA-50 group and virulence genes plcH, aprA, and algD were the important epidemiological characteristics of this group of P. aeruginosa. The present findings provide insights into the mechanisms of hypervirulence as well as resistance to β-lactams and aminoglycosides. To the best of our knowledge, this is the first report of blaPDC, blaOXA-50, and aph(3')-XV in P. aeruginosa in China.

Propagation of blaKPC-2 within two sequence types of Escherichia coli in a tertiary referral hospital of northeast India

KPC enzyme encoded by blaKPC gene has become a potential source of carbapenem resistance and a challenge to clinicians' worldwide and has been gradually emerging in India. The study investigated Escherichia coli isolates of clinical origin for the carriage of blaKPC-2 along with their resistance profile, carriers, co-existing resistance determinants and clonal types. E. coli isolates with non-susceptibility to ertapenem were collected and were investigated for the presence of blaKPC-2 and co-existence of other carbapenemases, ESBLs, aminoglycoside modifying enzymes genes, 16S methyltransferase genes, sulphonamides and quinolone resistance using PCR assay. Transformation and conjugation assay were performed to check horizontal transferability. Plasmid incompatibility typing and multilocus sequence typing was also performed. Two E. coli isolates belonging to two sequence types ST9582 and ST6171 were identified with the carriage of blaKPC-2. The isolates were multidrug resistant, however, were found were susceptible to colistin, nitrofurantoin and tigecycline. In addition to blaKPC-2, the isolate co-harboured a number of resistance genes including blaNDM-1, blaCTX-M-15, ant(2″)-Ia, aac(3)-I, aac(3)-IIc, aac(6′)-Ib and sul3. The conjugative Inc FIC plasmid was identified in our study with the carriage of blaKPC-2 gene in this study centre. The carriage of blaKPC-2 gene within conjugatively transferable Inc FIC plasmid warrants close monitoring to control inter and intra species spread.

Characteristics of High-Level Aminoglycoside-Resistant Enterococcus faecalis Isolated from Bulk Tank Milk in Korea.

Simple SummaryAminoglycosides are used to treat various infections in veterinary and human medicine. However, with the emergence of high-level aminoglycoside resistance in human and food-producing animals, the synergism of aminoglycosides with beta-lactam or glycopeptide is being threatened. Moreover, the environmental mastitis-causing agent, enterococci, has emerged as a cause of nosocomial infection due to its antimicrobial resistance. Therefore, the purpose of this study was to investigate the characteristics of high-level aminoglycoside-resistant Enterococcus faecalis isolated from bulk tank milk in Korea. It showed that 185 (61.5%) isolates out of 301 were high-level aminoglycoside resistant, while 149 isolates were multidrug resistant.Enterococci, which are considered environmental mastitis-causing pathogens, have easily acquired aminoglycoside-resistant genes that encode various aminoglycoside-modifying enzymes (AME). Therefore, this study was conducted to compare the distribution of high-level aminoglycoside-resistant (HLAR) and multidrug-resistant (MDR) Enterococcus faecalis (E. faecalis) bacteria isolated from bulk tank milk in four dairy companies in Korea. Moreover, it analyzed the characteristics of their antimicrobial resistance genes and virulence factors. Among the 301 E. faecalis bacteria studied, 185 (61.5%) showed HLAR with no significant differences among the dairy companies. Furthermore, 129 (69.7%) of the 185 HLAR E. faecalis showed MDR without significant differences among companies. In contrast, HLAR E. faecalis from companies A, B, and C were significantly higher in resistance to the four classes than those in company D, which had the highest MDR ability against the three antimicrobial classes (p < 0.05). In addition, in the distribution of AME genes, 72 (38.9%) and 36 (19.5%) of the isolates carried both aac(6′)Ie-aph(2″)-la and ant(6)-Ia genes, and the ant (6)-Ia gene alone, respectively, with significant differences among the companies (p < 0.05). In the distribution of virulence genes, the ace (99.5%), efa A (98.9%), and cad 1 (98.4%) genes were significantly prevalent (p < 0.05). Thus, our results support that an advanced management program by companies is required to minimize the dissemination of antimicrobial resistance and virulence factors.

Clonal change of carbapenem-resistant Acinetobacter baumannii isolates in a Korean hospital.

The expansion of specific carbapenem-resistant Acinetobacter baumannii (CRAB) clones is a global concern due to its therapeutic difficulty and epidemicity. To understand the prevalence of CRAB isolates in a Korean hospital, we investigated the epidemiological characteristics of 96 CRAB isolates between 2016 and 2018, including the sequence types (STs), antimicrobial susceptibility, and genetic background of resistance to carbapenems and aminoglycosides. Six STs were identified using the Oxford multilocus sequence typing scheme; ST191 (n=8), ST208 (n=12), ST229 (n=11), and ST369 (n=21) were previously identified clones in the study hospital, whereas gpi variants of ST208, ST451 (n=34) and ST784 (n=10), were emerging clones. ST208 isolates exhibited higher resistance rates to minocycline than other ST isolates, whereas ST369 isolates exhibited lower resistance rates to aminoglycosides and trimethoprim/sulfamethoxazole than other ST isolates. All CRAB isolates previously isolated in the study hospital carried ISAbaI-blaOXA-23 for carbapenem resistance, but 10 ST229 isolates carried only ISAbaI-blaOXA-51. The carriage of armA was lower in ST369 isolates (38%) than in other ST isolates (≥83%). The frequency and diversity of aminoglycoside-modifying enzyme genes were decreased among the CRAB isolates between 2016 and 2018 compared with CRAB isolates between 2013 and 2015 at the study hospital. In conclusion, clonal complex 208 CRAB isolates are predominant in the study hospital. This study demonstrates the evolutionary change of CRAB isolates in the study hospital in relation to the emergence of new STs and selection of resistant genes.

Coexistence of aminoglycoside resistance genes in CTX-M-producing isolates of Klebsiella pneumoniae in Bushehr province, Iran.

Background and Objectives:Increasing the rate of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae has given rise to a major healthcare issue in clinical settings over the past few years. Treatment of these strains is hardly effective since the plasmid encoding ESBL may also carry other resistance genes including aminoglycosides. The current study aimed to evaluate the prevalence of ESBL-producing K. pneumoniae and investigate the coexistence of Cefoxitamase-Munich (blaCTX-M) with aminoglycoside-modifying enzyme (AME) genes, aac(3)IIa as well as aac(6′)Ib, in CTX-M-producing K. pneumoniae isolated from patients in Bushehr province, Iran.Materials and Methods:A total of 212 K. pneumoniae isolates were collected and confirmed using polymerase chain reaction (PCR) of the malate dehydrogenase gene. Isolates were screened for production of ESBL. Phenotypic confirmatory test was performed using combined disk test. The genes encoding CTX-M groups and AME genes, aac(3)IIa and aac(6′)Ib, were investigated by PCR.Results:The ESBL phenotype was detected in 56 (26.4%) K. pneumoniae isolates. Moreover, 83.9% of ESBL-producing isolates carried the genes for CTX-M type β-lactamases, which were distributed into the two genetic groups of CTX-M-1 (97.8%)- and CTX-M-2 (2.1%)-related enzymes. Notably, among K. pneumoniae isolates containing the blaCTX-M gene, 68.08% of isolates harbored AME genes. In addition, the coexistence of blaCTX-M with aac(3)-IIa and aac(6′)-Ib was observed in 46.8% of CTX-M-producing K. pneumoniae isolates.Conclusion:This study provides evidence of a high prevalence of AME genes in CTX-M-producing K. pneumoniae isolates; therefore, in the initial empirical treatment of infections caused by ESBL-KP in regions with such antibiotic resistance patterns, aminoglycoside combination therapy should be undertaken carefully.

Prevalence of Aminoglycoside Resistance and Aminoglycoside Modifying Enzymes in Acinetobacter baumannii Among Intensive Care Unit Patients, Ismailia, Egypt

BackgroundAcinetobacter baumannii is an opportunistic pathogen that rapidly develops antibiotic resistance against commonly prescribed antimicrobial agents in hospitalized patients worldwide. Aminoglycosides are commonly used in the treatment of A. baumannii health care-associated infections (HAIs). Aminoglycosides resistance mechanisms are varied and commonly involve production of aminoglycoside-modifying enzymes (AME) and efflux systems.AimThis study aimed to provide an insight into the frequency of genes encoding AME in A. baumannii strains isolated from different clinical specimens in intensive care units (ICU).MethodologyA total of 52 multidrug-resistant (MDR) A. baumannii strains were isolated from ICU, Suez Canal University Hospitals. Species identification and antibiotics susceptibility testing were done by the automated system VITEK 2. The genes encoding AME were detected by PCR.ResultsAminoglycosides resistance (amikacin, gentamicin and tobramycin) was observed in 35 isolates (67.3%). We found that aacC1 gene was the predominant AME resistance gene among A. baumannii isolates, detected in 14 isolates (40%), aphA6 in 11 isolates (31.4%) and addA1 in 5 isolates (14.2%). We found 5 isolates containing 2 AME genes, 3 of them with aacC1 and aphA6 and the remaining 2 with both aacC1 and aadA1 genes. Nearly, 5 isolates (14.2%) were negative for all AME resistance genes.ConclusionOur study indicated that AME encoding genes are predominant in A. baumannii strains in our region which stressed on the importance of preventive measures to control spreading of resistance genes.

Investigation of isepamicin in-vitro efficiency in Gram negative bacteria

ContextIsepamicin is a new semisynthetic aminoglycoside derived from gentamicin B and it is effective against Gram negative bacteria. Antibiotic resistance is an emerging problem and new options need for the treatment of infections caused by Gram negative bacteria. AimsIn this study we aimed to investigate the in vitro efficiency in carbapenem susceptible and nonsusceptible Enterobacterales and Pseudomonas aeruginosa. Methods and materialA total of 214 isolates of Gram-negative bacteria (Enterobacterales n = 129 and P. aeruginosa n = 85). Identification of the bacteria was tested in Vitek MS (Biomeriux, France). Susceptibility of isepamicin, amikacin, gentamicin, tobramycin and netilmicin was determined by Kirby Bauer disc diffusion method. The breakpoints for susceptibility to isepamicin, amikacin, gentamicin, streptomycin, tobramycin and netilmicin were evaluated according to the Comité de l’Antibiogramme dela Société Française de Microbiologie (CA-SFM) and EUCAST, respectively. Aminoglycoside modifying enzyme (AME) genes were investigated by multiplex PCR method. ResultsIsepamicin susceptibility was determined as 92.3% for Enterobacterales and 67% for P. aeruginosa and 94.4% for carbapenem resistant Enterobacterales. The most common AME gene was aac (6′)-Ib in both Enterobacterales (76%) and P. aeruginosa (14.1%). Seven of the isepamicin intermediate or resistant isolates were positive aac (6’)-Ib in Enterobacterales and P. aeruginosa. ConclusionsIn this study, isepamicin showed good efficiency against both susceptible and carbapenem nonsusceptible Enterobacterales. But amikacin was prior to isepamicin P. aeruginosa isolates. Isepamicin could be a therapeutic option for the infections caused by Enterobacterales.

Role of aminoglycoside-modifying enzymes and 16S rRNA methylase (ArmA) in resistance of Acinetobacter baumannii clinical isolates against aminoglycosides.

INTRODUCTION: This study aimed to determine the role of genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylase (ArmA) in Acinetobacter baumannii clinical isolates. METHODS: We collected 100 clinical isolates of A. baumannii and identified and confirmed them using microbiological tests and assessment of the OXA-51 gene. Antibiotic susceptibility testing was carried out using disk agar diffusion and micro-broth dilution methods. The presence of AME genes and ArmA was detected by PCR and multiplex PCR. RESULTS: The most and least effective antibiotics in this study were netilmicin and ciprofloxacin with 68% and 100% resistance rates, respectively. According to the minimum inhibitory concentration test, 94% of the isolates were resistant to gentamicin, tobramycin, and streptomycin, while the highest susceptibility (20%) was observed against netilmicin. The proportion of strains harboring the aminoglycoside resistance genes was as follows: APH(3′)-VIa (aphA6) (77%), ANT(2”)-Ia (aadB) (73%), ANT(3”)-Ia (aadA1) (33%), AAC(6′)-Ib (aacA4) (33%), ArmA (22%), and AAC(3)-IIa (aacC2) (19%). Among the 22 gene profiles detected in this study, the most prevalent profiles included APH(3′)-VIa + ANT(2”)-Ia (39 isolates, 100% of which were kanamycin-resistant), and AAC(3)-IIa + AAC(6′)-Ib + ANT(3”)-Ia + APH(3′)-VIa + ANT(2”)-Ia (14 isolates, all of which were resistant to gentamicin, kanamycin, and streptomycin). CONCLUSIONS: High minimum inhibitory concentration of aminoglycosides in isolates with the simultaneous presence of AME- and ArmA-encoding genes indicated the importance of these genes in resistance to aminoglycosides. However, control of their spread could be effective in the treatment of infections caused by A. baumannii.

Optimizing aminoglycoside selection for KPC-producing Klebsiella pneumoniae with the aminoglycoside-modifying enzyme (AME) gene aac(6')-Ib.

KPC-producing Klebsiella pneumoniae (KPC-Kp) isolates commonly co-harbour the aminoglycoside-modifying enzyme (AME) gene aac(6')-Ib, which encodes an AME that can confer resistance to some of the commercially available aminoglycosides. We sought to determine the influence of AAC(6')-Ib in KPC-Kp on the pharmacodynamic activity of aminoglycosides. Six KPC-Kp clinical isolates, three with and three without aac(6')-Ib, were analysed. Using these isolates, the bacterial killing of amikacin, gentamicin and tobramycin was assessed in static time-kill experiments. The pharmacodynamic activity of the aminoglycosides was then assessed in a dynamic one-compartment infection model over 72 h using simulated human pharmacokinetics of once-daily dosing with amikacin (15 mg/kg), gentamicin (5 mg/kg) and tobramycin (5 mg/kg). At clinically relevant aminoglycoside concentrations in time-kill experiments and the dynamic one-compartment model, gentamicin was more active than amikacin or tobramycin against the isolates harbouring aac(6')-Ib. Amikacin, gentamicin and tobramycin all showed progressively reduced bacterial killing with exposure to repeated doses against most isolates in the dynamic one-compartment model. MIC values were generally not a good predictor of gentamicin pharmacodynamic activity against KPC-Kp, but were more reliable for amikacin and tobramycin. Gentamicin may be preferred over amikacin or tobramycin for treatment of KPC-Kp infections. However, gentamicin MICs are not a consistent predictor of its pharmacodynamic activity and unexpected treatment failures are possible.

Monitoring of Non-β-Lactam Antibiotic Resistance-Associated Genes in ESBL Producing Enterobacterales Isolates.

Genetic context of extended spectrum β-Lactamase (ESBL) producing Enterobacterales and its association with plasmid mediated quinolone resistance (PMQR), aminoglycoside modifying enzymes (AME) and Trimethoprim/Sulfamethoxazole (TMP-SMX) resistance is little known from North India. Therefore, the current study was aimed to investigate the frequency of Non-β-Lactam antibiotic resistance associated genes in extended spectrum β-Lactamase producing Enterobacterales. For this study, Non-Duplicate phenotypically confirmed ESBL producing Enterobacterales isolates (N = 186) were analyzed for ESBLs, PMQRs, AMEs and TMP-SMX resistance genes using polymerase chain reaction (PCR). PCR detected presence of PMQR genes in 81.29% (N = 139) of ESBL isolates (N = 171), AME genes in 60.82% and TMP-SMX resistance genes in 63.74% of the isolates. Molecular characterization of ESBL producing Enterobacterales showed 84.79% blaTEM followed by 73.68% blaCTX-M, 43.86% blaSHV, 19.88% blaPER and 9.94% blaVEB, respectively. Analysis of PMQR genes revealed 77.7% aac(6′)-lb-cr the most commonly detected gene followed by 67.63% oqxB, 62.59% oqxA, 43.17% qnrB, 19.42% qnrD, 18.7% qnrS, 9.35% qnrA, 3.6% qepA and 2.88% qnrC, respectively. Analysis of AMEs gene profile demonstrated 81.73% aac(6′)-Ib, the most frequently encountered gene followed by 46.15% aph(3′)-Ia, 44.23% ant(3”)-Ia, respectively. A 100% prevalence of sul1, followed by dfrA (54.63%) and sul2 (15.74%) was observed. In summary, prevalence of ESBL-Producing genes (particularly blaTEM and blaCTX-M) along with PMQR, AMEs, and TMP-SMX resistant genes may potentially aid in the transfer of antimicrobial resistance among these strains.

Genetic diversity and prevalence of aminoglycoside modifying enzymes among Escherichia coli strains isolated from inpatients with urinary tract infections

The aminoglycosides are broad-spectrum antibiotics that traditionally prescribed for the treatment of serious Gram-negative infections. The main mechanism of resistance to these antibiotics in Escherichia coli is enzymatic alteration of aminoglycosides by aminoglycoside-modifying enzymes (AMEs). The present study aimed to investigate the genetic diversity and prevalence of aminoglycoside-resistant strains of E. coli isolated from inpatients with urinary tract infection. This cross-sectional study was conducted during September 2018 to March 2019 on a total of 83 non-susceptible aminoglycosides E. coli in the North of Iran. Antimicrobial susceptibility test was performed by disk diffusion method. The presence of genes encoding aminoglycoside modifying enzymes (AMEs) was detected by PCR. The clonal relatedness of E. coli isolates was investigated by ERIC-PCR. Totally, 66.3% and 43.4% of isolates were non-susceptible to gentamicin and amikacin, respectively. The most common AME gene was aac(6′)-Ib (49.4%) followed by aac(3)-II (39.8%), ant(3″)-I (8.4%) and aph(3′)-VI (1.2%). Moreover, aac(6′)-II gene was not found among studied strains. All of the extended-spectrum beta-lactamases (ESBLs) producing isolates (55 isolates) were tested by ERIC-PCR. These isolates were classified into 10 ERIC types, and ERIC type A were the predominate type containing 52.7% of isolates. Findings showed a substantial proportion of AMEs among aminoglycosides-resistant UPEC isolates. Moreover, the clonal analysis showed that a remarkable rate (52.7%) of aminoglycosides-resistant and ESBL-producing isolates was closely related. These findings suggest the necessity of restricted infection control policy to prevent further dissemination of resistant strains.

Distribution of Staphylococcal cassette chromosome mecA (SCCmec) types among coagulase-negative Staphylococci isolates from healthcare workers in the North-West of Iran.

Objective(s):Methicillin-resistant coagulase-negative Staphylococci (MR-CoNS) are recognized as one of the major causes of healthcare-associated infections in hospitals. The present investigation aimed to study the prevalence of Staphylococcal cassette chromosome mec (SCCmec) types, along with aminoglycoside modifying enzymes (AMEs) genes in the nasal carriage of MR-CoNS in the north-west of Iran.Materials and Methods:To assess the potential of coagulase-negative Staphylococci as hidden reservoirs for antibiotic resistance, we analyzed the antimicrobial susceptibility of MR-CoNS using the disk diffusion method. In addition, PCR and multiplex PCR assays were performed to determine the prevalence of AME encoding genes and SCCmec types in methicillin-resistant coagulase-negative Staphylococci isolates.Results:A total of 51 MR-CoNS isolates were recovered from the anterior nares of healthcare workers. The observed resistance rates to tobramycin, gentamicin, cotrimoxazole, kanamycin, erythromycin, tetracycline, and ciprofloxacin were 74.5%, 68.5%, 57%, 53%, 51%, 49%, and 8%, respectively. Of the 51 tested MR-CoNS isolates, 2(4%) were harboring SCCmec type I, four (8%) were type II, six (12%) type III, eleven (21.6%) type IVa, two (4%) type IVb, two (4%) type IVc, six (12%) type IVd, and two (4%) type V. The rates of prevalence of the aminoglycoside modifying enzyme genes were as follows: aac (6′)/aph (2′′) (28 cases, 55 %), ant (4′)-Ia (20 cases, 39%), and the aph (3´)-IIIa gene (9 cases, 17.6 %).Conclusion:Subtypes IVa and IVd were the most prevalent SCC elements, and aac (6′)/aph (2′′) was the most common AME gene detected among the MR-CoNS isolates.