16S rRNA Methylase Research Articles

Molecular Evaluation of Aminoglycoside Resistance and Biofilm Formation Potential in Escherichia coli Isolates Collected from Hospitalized Patients

Background: Resistance to antibiotics and the ability to develop biofilms, two main virulence determinants of Escherichia coli, play a crucial role in the persistence of infections. Objectives: The aim of this study was to evaluate aminoglycoside resistance and biofilm formation potential in E. coli isolates collected from hospitalized patients in the Southwest of Iran. Methods: A total of 70 E. coli clinical isolates from different specimens were collected from Ahvaz teaching hospitals affiliated with Ahvaz Jundishapur University of Medical Sciences. All the isolates were identified as E. coli using conventional microbiological tests. Susceptibility to antibiotics was determined using the Kirby–Bauer disk diffusion method. Biofilm formation was assessed using the microtiter plate method. Finally, PCR was conducted to detect virulence gene determinants, including fimbrial genes, aminoglycoside modifying enzymes (AMEs), and 16S rRNA methylase (RMTase) genes. Results: Among aminoglycoside antibiotics, E. coli isolates showed the highest and lowest resistance rates to tobramycin (TOB; 51.4%) and gentamicin (GEN; 24.2%), respectively. Simultaneous resistance to GEN, amikacin, and TOB was observed in 28.5% of the isolates, representing the most common antibiotic resistance pattern. The prevalence of strong biofilm producers was higher in the extensively drug-resistant (XDR) phenotype group compared to the multiple drug-resistant (MDR) group (76.1% vs. 23.8%). Among the 36 isolates resistant to at least one of the aminoglycoside antibiotics, 36.1% had AME-related genes, either alone or in various combinations. Most isolates harboring AME genes were also positive for the presence of biofilm-related genes, including ecpA and fimA. Conclusions: The most frequent AME-related genes were ant(2”)-Ia and aph(3’)-Ia, followed by aac(3’)-IIa. The findings of the present study provide probable evidence that GEN is an effective aminoglycoside against biofilm-producing and antibiotic-resistant E. coli isolates.

Prevalence and mechanisms of aminoglycoside resistance among drug-resistant Pseudomonas aeruginosa clinical isolates in Iran

BackgroundAminoglycosides have been a cornerstone of the treatment of nosocomial infections caused by Pseudomonas aeruginosa for over 80 years. However, escalating emergence of resistance poses a significant challenge. Therefore, this study aimed to investigate the prevailing patterns of aminoglycoside resistance among clinical isolates of P. aeruginosa in Iran; as well as the underlying resistance mechanisms observed in patients referred to Ardabil hospitals.MethodsA total of 200 isolates from five hospitals were evaluated. The resistance profiles of P. aeruginosa isolates to tobramycin, amikacin, and netilmicin were determined using the disk diffusion method. The capacity of aminoglycoside-resistant isolates to form biofilms was assessed through a phenotypic assay, and the results were confirmed using the gene amplification technique. The presence of genes associated with aminoglycoside resistance was detected using polymerase chain reaction (PCR). Quantitative reverse transcription PCR (qRT-PCR) was performed to measure the expression levels of genes encoding the MexXY-OprM efflux pump and PhoPQ two-component system (TCS).ResultsThe prevalence of aminoglycoside-resistant P. aeruginosa isolates was 48%, with 94.7% demonstrating multidrug resistance (MDR). All aminoglycoside-resistant P. aeruginosa strains exhibited biofilm-forming capabilities and harbored all the genes associated with biofilm production. Among the nine genes encoding 16S rRNA methylase and aminoglycoside-modifying enzymes, three genes were detected in these isolates: aac(6’)-Ib (85.4%), ant(2’’)-Ia (18.7%), and aph(3’)-VI (3.1%). Additionally, all aminoglycoside-resistant P. aeruginosa isolates carried mexY and phoP genes, although the expression levels of mexY and phoP were 75% and 87.5%, respectively.ConclusionGiven the considerably high prevalence of aminoglycoside-resistant P. aeruginosa strains, urgent measures are warranted to transition towards the use of novel aminoglycosides and to uphold vigilant surveillance of resistance patterns.

Occurrence and characterization of rmtB-harbouring Salmonella and Escherichia coli isolates from a pig farm in the UK.

To characterize and elucidate the spread of amikacin-resistant Enterobacteriaceae isolates from environmental samples on a pig farm in the UK, following the previous identification of index Salmonella isolates harbouring the rmtB gene, a 16S rRNA methylase. Environmental samples were collected during two visits to a pig farm in the UK. Isolates were recovered using selective media (amikacin 128 mg/L) followed by real-time PCR and WGS to analyse rmtB-carrying Salmonella and Escherichia coli isolates. Salmonella and E. coli isolates harbouring the rmtB gene were detected at both farm visits. All Salmonella isolates were found to be monophasic S. enterica serovar Typhimurium variant Copenhagen of ST34. rmtB-harbouring E. coli isolates were found to be one of three STs: ST4089, ST1684 and ST34. Long-read sequencing identified the rmtB gene to be chromosomally located in Salmonella isolates and on IncFII-type plasmids in E. coli isolates. The results showed the rmtB gene to be flanked by IS26 elements and several resistance genes. We report on the occurrence of rmtB-harbouring Enterobacteriaceae on a pig farm in the UK. rmtB confers resistance to multiple aminoglycosides and this work highlights the need for surveillance to assess dissemination and risk.

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.

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.

Klebsiella pneumoniae co-harbouring bla NDM-1 , armA and mcr-10 isolated from blood samples in Myanmar.

Background. The spread of Enterobacteriaceae coproducing carbapenemases, 16S rRNA methylase and mobile colistin resistance proteins (MCRs) has become a serious public health problem worldwide. This study describes two clinical isolates of Klebsiella pneumoniae coharbouring bla IMP-1, armA and mcr-10.Methods. Two clinical isolates of K. pneumoniae resistant to carbapenems and aminoglycosides were obtained from two patients at a hospital in Myanmar. Their minimum inhibitory concentrations (MICs) were determined by broth microdilution methods. The whole-genome sequences were determined by MiSeq and MinION methods. Drug-resistant factors and their genomic environments were determined.Results. The two K. pneumoniae isolates showed MICs of ≥4 and ≥1024 µg ml-1 for carbapenems and aminoglycosides, respectively. Two K. pneumonaie harbouring mcr-10 were susceptible to colistin, with MICs of ≤0.015 µg ml-1 using cation-adjusted Mueller-Hinton broth, but those for colistin were significantly higher (0.5 and 4 µg ml-1) using brain heart infusion medium. Whole-genome analysis revealed that these isolates coharboured bla NDM-1, armA and mcr-10. These two isolates showed low MICs of 0.25 µg ml-1 for colistin. Genome analysis revealed that both bla NDM-1 and armA were located on IncFIIs plasmids of similar size (81 kb). The mcr-10 was located on IncM2 plasmids of sizes 220 or 313 kb in each isolate. These two isolates did not possess a qseBC gene encoding a two-component system, which is thought to regulate the expression of mcr genes.Conclusion. This is the first report of isolates of K. pneumoniae coharbouring bla NDM-1, armA and mcr-10 obtained in Myanmar.

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.

Molecular evaluation of aminoglycosides resistance and biofilm formation in Klebsiella pneumoniae clinical isolates: A cross-sectional study.

Resistance to antibiotics and the capability to develop biofilm as two main virulent determinants of Klebsiella pneumoniae have important role in infection persistence. The aim of the study was to evaluate the association between the prevalence of aminoglycoside resistance and virulence genes and biofilm formation capacity in K. pneumoniae strains isolated from hospitalized patients in South-West of Iran. A total of 114 non-duplicate clinical isolates of K. pneumoniae collected from Ahvaz teaching hospitals. Identification of species was performed by biochemical tests and then confirmed by polymerase chain reaction(PCR) of rpoB gene. The susceptibility to antibiotics was determined by Kirby-Bauer disk diffusion method. Biofilm formation was assessed by microtiter plate method. Finally, PCR was conducted to detect virulence gene determinants including fimbrial genes, aminoglycoside modifying enzymes- and 16S rRNA methylase (RMTase) genes. Totally, all collected strains were carbapenem resistant and showed multidrug- and extensively drug-resistance phenotype (75% and 25%, respectively). Seventy-one percent (n = 81) of isolates were non-susceptible to aminoglycosides. Among aminoglycoside antibiotics, K. pneumoniae isolates showed the highest and lowest resistance rates to tobramycin (71%) and the amikacin (25%), respectively. All biofilm producer strains were positive for the presence virulence determinants including ecpA, fimA, mrkD, and mrkA. Of 81 aminoglycosides non-susceptible isolates 33% were positive for the presence ant (2″)-Ia as the most prevalent gene followed by aac (3')-IIa and armA (27%), aac (6')-Ib (18%), and aph (3')-Ia (15%). K. pneumoniae isolates showed the highest and the lowest aminoglycoside resistance rates to tobramycin and amikacin, respectively. Majority of isolates were biofilm producers and there was significant association between antibiotic resistance pattern and the strength of biofilm production. The ant(2″)-Ia, aac (3')-IIa, and armA genes in aminoglycoside-resistant isolates.

Molecular patterns of clinically important fluoroquinolone resistance in multidrug-resistant Klebsiella pneumoniae isolates during nosocomial outbreaks in Shanghai, PR China.

Introduction. The soaring resistance of Klebsiella pneumoniae to fluoroquinolones in PR China has substantially limited the application of these antimicrobials, especially in those clinical settings that were threatened by persistent carbapenem-resistant K. pneumoniae (CRKP), necessitating strict implementation of antimicrobial stewardship and active enhanced surveillance of infection control.Hypothesis. There is interplay between plasmid-mediated quinolone resistance (PMQR) determinants and quinolone resistance-determining region (QRDR) mutations during the acquisition of a clinically important fluoroquinolone resistance (CI-FR) profile in multidrug-resistant K. pneumoniae (MDR-KP) isolates.Aim. To investigate the high-risk CRKP clones responsible for nosocomial spread and analyse the molecular patterns of CI-FR in MDR-KP isolates in a tertiary hospital in Shanghai, PR China.Methodology. A total of 34 isolates, including 30 CRKPs, were molecularly characterized. Investigations included antimicrobial susceptibility tests, multilocus sequence typing (MLST) and wzi genotyping, PCR sequencing and phylogenetic analysis for resistance-associated genes, and clinical information retrieval from medical records.Results. Two high-risk CRKP clones, ST11-wzi64 and ST15-wzi19/wzi24, were identified as being responsible for nosocomial outbreaks in the intensive care unit (ICU) and the neurosurgery department, potentially by the respiratory route. QRDR mutations of both gyrA and parC were detected in isolates of ST15 (S83F/D87A/S80I), ST11 (S83I/D87G/S80I) and ST218 (D87A/S80I), respectively. The PMQR genes, qnrS1, aac(6')-Ib-cr and oqxAB, were present in 32 (94.1 %) of the isolates alone or in combination, co-occurring with genes (bla) encoding β-lactamases, 16S rRNA methylases and putrescine ABC permeases. AcrR, an AcrAB transcriptional repressor, was insertion-inactivated by the IS5-like element in ST11 isolates. The encoding sequences of OmpK35 and OmpK36 genes were associated with specific STs and wzi alleles. ST11, ST15-wzi19 and ST218 isolates had frameshift disruptions in OmpK35 and specific GD insertions at position 134-135 in OmpK36. The 27 isolates with clinically important ciprofloxacin resistance (MICs ≥2 mg l-1) included 25 isolates (ST15, ST11, ST218) with multiple QRDR mutations, plus 1 with only 2 PMQR determinants (ST290-wzi21) and another with an unknown resistance mechanism (ST65-wzi72). Ciprofloxacin-susceptible isolates maintained intact ompK36 genes, including two CRKPs each with ST13-wzi74 (KPC-2 and NDM-1 coproducers) and ST65-wzi72, plus carbapenem-susceptible isolates (ST15-wzi24, ST65-wzi72, ST107-wzi173).Conclusions. Under selective pressures, the accumulation of mutations of three types (QRDR, acrR, ompK36) and the acquisition of resistance-conferring genes have continuously contributed to CI-FR in MDR-KP isolates.

Intra- and Interspecies Spread of a Novel Conjugative Multidrug Resistance IncC Plasmid Coharboring blaOXA-181 and armA in a Cystic Fibrosis Patient.

ABSTRACTA novel multidrug resistance conjugative 177,859-bp IncC plasmid pJEF1-OXA-181 coharboring the carbapenemase-coding blaOXA181 and the aminoglycoside resistance 16S rRNA methyltransferase-coding armA genes was detected in two unrelated Escherichia coli gut isolates of ST196 and ST648, as well as two ST35 Klebsiella pneumoniae gut and sputum isolates of a cystic fibrosis patient. The armA gene was located within the antimicrobial resistance island ARI-A and the blaOXA181 gene, which was preceded by IS903 and ISEcp1Δ was inserted within the transfer genes region without affecting conjugation ability. Comparative plasmid analysis with other related IncC plasmids showed the presence of blaOXA181, as well as its integration site, are thus far unique for these types of plasmids. This study illustrates the potential of a promiscuous multidrug resistance plasmid to acquire antibiotic resistance genes and to disseminate in the gut of the same host.IMPORTANCE Colocalization of carbapenemases and aminoglycoside resistance 16S rRNA methylases on a multidrug resistance conjugative plasmid poses a serious threat to public health. Here, we describe the novel IncC plasmid pJEF1-OXA-181 cocarrying blaOXA-181 and armA as well as several other antimicrobial resistance genes (ARGs) in different Enterobacterales isolates of the sputum and gut microbiota of a cystic fibrosis patient. IncC plasmids are conjugative, promiscuous elements which can incorporate accessory antimicrobial resistance islands making them key players in ARGs spread. This plasmid was thus far unique among IncC plasmids to contain a blaOXA-181 which was integrated in the transfer gene region without affecting its conjugation ability. This study highlights that new plasmids may be introduced into a hospital through different species hosted in one single patient. It further emphasizes the need of continuous surveillance of multidrug-resistant bacteria in patients at risk to avoid spread of such plasmids in the health care system.

Emergence and Transfer of Plasmid-Harbored rmtB in a Clinical Multidrug-Resistant Pseudomonas aeruginosa Strain.

Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a great challenge to clinical treatment. In this study, we characterized a ST768 MDR P. aeruginosa strain, Pa150, that was isolated from a diabetic foot patient. The minimum inhibitory concentration (MIC) assay showed that Pa150 was resistant to almost all kinds of antibiotics, especially aminoglycosides. Whole genome sequencing revealed multiple antibiotic resistant genes on the chromosome and a 437-Kb plasmid (named pTJPa150) that harbors conjugation-related genes. A conjugation assay verified its self-transmissibility. On the pTJPa150 plasmid, we identified a 16S rRNA methylase gene, rmtB, that is flanked by mobile genetic elements (MGEs). The transfer of the pTJPa150 plasmid or the cloning of the rmtB gene into the reference strain, PAO1, significantly increased the bacterial resistance to aminoglycoside antibiotics. To the best of our knowledge, this is the first report of an rmtB-carrying conjugative plasmid isolated from P. aeruginosa, revealing a novel possible transmission mechanism of the rmtB gene.

Antimicrobial Resistance Analysis of Escherichia coli Isolated from Pigeons in Qingdao, Shandong Province, China.

With the development of the pigeon industry in Qingdao, more attention is paid to scientific breeding and precise treatment. This study isolated and identified Escherichia coli from pigeons in Qingdao to determine their susceptibility to 18 antibiotics. The PCR method was used to detect the prevalence of Extended-Spectrum β-Lactamase genes, carbapenem resistance genes, 16S rRNA methylase genes and plasmid-mediated colistin resistance genes in the isolates. The results showed that Escherichia coli isolated from pigeons in Qingdao were the most resistant to tetracycline, followed by ampicillin, conventional cyclosporines, quinolones, cephalosporins, and aminoglycosides. No isolates were found to be resistant to amikacin, meropenem, colistin, tigecycline, and fosfomycin. The resistance to some antibiotics (ampicillin, tetracycline, and florfenicol) and the muti-drug resistance of Escherichia coli from meat pigeons were both higher than those from homing and ornamental pigeons. A total of 24.8% of the isolates showed multi-drug resistance, especially triple-drug resistance. Two isolates were found to carry ESBLs resistance genes. Hopefully, this study will provide a certain scientific basis for the clinical medication of pigeon colibacillosis, helping to prevent antimicrobial resistance transmission of Escherichia coli among different host animals and humans and maintain public health safety in Qingdao.

Spread of armA 16S rRNA Methylase Gene in a Tertiary Hospital and Errors in Aminoglycoside Susceptibility Results of Rapid Susceptibility Test Systems

Aminoglycosides (AGs) are actively used in combination therapies against carbapenem resistant gram negative species in recent years. Spread of 16S rRNA methylases which can cause high-level resistance to AG antibiotics, limits this treatment choice. Although there are some studies showing that errors in determining AG susceptibility in automated systems may be related to the armA gene, one of the 16S rRNA methylase genes, the exact reason for these errors is not yet known. In our study, we aimed to investigate the relevance of 16S rRNA methylases to the discrepancies between VITEK 2.0 and disc diffusion test results for amikacin (AK) and gentamicin (GEN) susceptibility of Acinetobacter baumannii and Klebsiella pneumoniae isolates. All K.pneumoniae and A.baumannii isolates from 1st January-10th February 2018 were collected prospectively and included in the study. Additionally, two initial isolates from July 2017 (one K.pneumoniae and one A.baumannii isolate) for which first discrepant susceptibility results were determined, were also included. Amikacin and gentamicin susceptibility results of 37 isolates [A. baumannii (n= 20) and K.pneumoniae (n= 17)] were evaluated together with VITEK 2.0 system, disc diffusion and gold standard broth microdilution methods and minor error (mE), major error (ME) and very major error (VME) rates were calculated. The rmtB, rmtC and armA genes in isolates were investigated by polymerase chain reaction (PCR) and the relationship between the presence of 16S rRNA methylases and false susceptibility results were examined. In addition, disc diffusion test results were evaluated at the end of four, six, eight hours and one night incubation periods to examine the effect of the double zone phenotype observed in 13 of the study isolates on rapid susceptibility tests. All disc diffusion test results were found to be compatible with broth microdilution test results. When the VITEK 2.0 system and the broth microdilution test were compared, 10.3% and 12.1% VME and 8.1% and 5.4% mE were detected for AK and GEN susceptibility results, respectively. While rmtB and rmtC genes were not detected in the study isolates, armA gene was positive in eight (47.1%) of 17 K.pneumoniae isolates and in 15 (75%) of 20 A.baumannii isolates. All three VMEs in A.baumannii isolates were detected in AK susceptibility results. Two of those were armA gene positive and one was armA gene negative isolates. All four VMEs in K. pneumoniae isolates were detected in GEN susceptibility results only, and all of these isolates were armA gene positive. No direct correlation was found between the errors detected in the VITEK 2.0 system susceptibility results and the double zone phenotype. When the isolates were evaluated in the 4-16 hours incubation time interval, it was observed that resistant colonies could be detected after a minimum of six hours of incubation period in the inhibition zone surrounding the aminoglycoside discs. To the best of our knowledge this is the first report of armA producing A.baumannii from Turkey. The high rate of armA gene positivity detected in our isolates suggested that the prevalence of armA gene increased in our country or at least in our region, in recent years. In the AG susceptibility results of the VITEK 2.0 system, the rate of VME above the acceptance criterion has shown that the errors occurred were not directly related to armA gene positivity or double zone phenotype. Finally, our study results indicated that AG susceptibility results should be evaluated minimum six hours later of incubation while implementing rapid susceptibility tests.

Spread of Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates Producing NDM-Type Metallo-β-Lactamase in Myanmar.

ABSTRACTA total of 38 isolates of carbapenem-resistant Klebsiella pneumoniae harboring blaNDM were obtained during surveillance of 10 hospitals in Myanmar. Of these 38 isolates, 19 (50%) harbored genes encoding 16S rRNA methylases, such as armA or rmtB. The K. pneumoniae strains tested belonged to 17 sequence types (STs), including the high-risk clonal lineages ST101 and ST147. The ST101 and ST147 isolates carried IncFII plasmids harboring blaNDM-5 and IncFIB(pQil) plasmids harboring blaNDM-1, respectively. These results indicate that IncFII plasmids harboring blaNDM-5 and IncFIB(pQil) plasmids harboring blaNDM-1 have been spreading in K. pneumoniae ST101 and ST147 isolates, respectively, in Myanmar.IMPORTANCE The emergence of carbapenem-resistant K. pneumoniae has become a serious problem in medical settings worldwide. The present study demonstrated that carbapenem-resistant K. pneumoniae strains have been spreading in medical settings in Myanmar. In particular, plasmid genes encoding NDMs and 16S rRNA methylases have been spreading in K. pneumoniae high-risk clones.

Clonal spread of ArmA- and OXA-23-coproducing Acinetobacter baumannii International Clone 2 in Brazil during the first wave of the COVID-19 pandemic.

Introduction. Carbapenem-resistant Acinetobacter baumannii (CRAB) is the primary pathogen causing hospital-acquired infections. The spread of CRAB is mainly driven by the dissemination of resistant clones, and in Latin America, International Clones IC-1 (also known as clonal complex CC1), IC-4 (CC15) and IC-5 (CC79) are the most prevalent.Gap Statement. There are no documented outbreaks of CRAB International Clone 2 (IC-2) reported in Brazil.Aim. To describe a large outbreak of CRAB caused by the uncommon IC-2 in a Brazilian COVID-19 hospital.Methodology. From May 2020 to May 2021, 224 patients infected or colonized with CRAB were identified in a single hospital; 92 % of them were also infected with SARS-CoV-2. From these patients, 137 isolates were recovered and subjected to antimicrobial susceptibility testing, PCR analysis and molecular typing. Whole-genome sequencing and downstream analysis were carried out on a representative isolate (the first available isolate).Results. In 76 % of the patients, a single OXA-23-producing CRAB IC-2 was identified. All the isolates were susceptible to polymyxin B, but highly resistant (>95 %) to aminoglycosides, fluoroquinolones and beta-lactams. Genomic analysis revealed that the representative isolate also carried the 16S rRNA Methylase ArmA, which was detected for the first time in this species in Brazil.Conclusion. We report the rapid spread of an emerging CRAB clone responsible for causing a large outbreak in a hospital in Brazil, a country with predominance of other CRAB clones. Continuous and prospective surveillance is warranted to evaluate the impact of this clone in Brazilian hospital settings.

Increasing Trends of Association of 16S rRNA Methylases and Carbapenemases in Enterobacterales Clinical Isolates from Switzerland, 2017-2020.

Aminoglycosides (AGs) in combination with β-lactams play an important role in antimicrobial therapy in severe infections. Pan-resistance to clinically relevant AGs increasingly arises from the production of 16S rRNA methylases (RMTases) that are mostly encoded by plasmids in Gram-negative bacteria. The recent emergence and spread of isolates encoding RMTases is worrisome, considering that they often co-produce extended-spectrum β-lactamases (ESBLs) or carbapenemases. Our study aimed to retrospectively analyze and characterize the association of carbapenem- and aminoglycoside-resistant clinical isolates in Switzerland during a 3.5-year period between January 2017 and June 2020. A total of 103 pan-aminoglycoside- and carbapenem-resistant clinical isolates were recovered at the NARA (Swiss National Reference Center for Emerging Antibiotic Resistance) during the 2017–2020 period. Carbapenemase and RMTase determinants were identified by PCR and sequencing. The characterization of plasmids bearing resistance determinants was performed by a mating-out assay followed by PCR-based replicon typing (PBRT). Clonality of the isolates was investigated by multilocus sequence typing (MLST). Over the 991 Enterobacterales collected at the NARA during this period, 103 (10.4%) of them were resistant to both carbapenems and all aminoglycosides. Among these 103 isolates, 35 isolates produced NDM-like carbapenemases, followed by OXA-48-like (n = 23), KPC-like (n = 21), or no carbapenemase (n = 13), OXA-48-like and NDM-like co-production (n = 7), and VIM-like enzymes (n = 4). The RMTases ArmA, RmtB, RmtC, RmtF, RmtG, and RmtB + RmtF were identified among 51.4%, 13.6%, 4.9%, 24.3%, 1%, and 1%, respectively. Plasmid co-localization of the carbapenemase and the RMTase encoding genes was found among ca. 20% of the isolates. A high diversity was identified in terms of the nature of associations between RMTase and carbapenemase-encoding genes, of incompatibility groups of the corresponding plasmids, and of strain genetic backgrounds, highlighting heterogeneous importations rather than clonal dissemination.

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.

Characterization of Aminoglycoside Modifying Enzymes Producing E. coli and Klebsiella pneumoniae Clinical Isolates

Antimicrobial resistance gene profile characterization and dissemination offer useful detail on the possible challenge in treating bacteria. The development of aminoglycoside modifying enzymes (AMEs) is considered as the primary mechanism of resistance to aminoglycosides, in addition to the 16S rRNA methylases. This study aimed at isolation and characterization of aminoglycosides resistant clinical isolates of enterobacteriaceae family from different clinical samples. Over a period of 24 months, thirty samples were collected and 49 clinical isolates of E. coli [n=25], Klebsiella [n=13], Enterobacter species (n=7) and Proteus species (n=4) were isolated from Egyptian clinical laboratories. The identities of the cultures were confirmed following standard microbiological procedures. Resistance of the isolates to aminoglycosides was determined by the disc diffusion method and isolates with highest resistance (n=9) were selected and investigated for 16S rRNA methylase and AMES encoding genes by polymerase chain reaction (PCR) and sequencing. In general, aminoglycoside resistance was found in 95% of the isolates; the isolates displayed the highest rate of resistance to netilmicin (75%) and kanamycin (55%), while resistance to gentamycin (18%) and tobramycin (16%) was low. A total of 9 isolates have the highest aminoglycoside resistant rate, showed the highest appearance for aac(6′)-Ib as well as ant (3″)-Ia resistant genes, with aac (3)-II (44%) and ant (4′)-IIb (34%) following closely. The high prevalence of AMEs observed among resistant isolates in this study suggests the urgent need for more efficient treatment designs to mitigate the selection burden as well as improved care of patients who have been infected with these drug-resistant organisms.

A Patient With Multiple Carbapenemase Producers Including an Unusual Citrobacter sedlakii Hosting an IncC bla NDM-1- and armA-carrying Plasmid.

Background.Patients colonized with multiple species of carbapenemase-producing Enterobacterales (CPE) are increasingly observed. This phenomenon can be due to the high local prevalence of these pathogens, the presence of important host risk factors, and the great genetic promiscuity of some carbapenemase genes.Methods.We analyzed 4 CPE (Escherichia coli, Klebsiella pneumoniae, Providencia stuartii, Citrobacter sedlakii), 1 extended-spectrum cephalosporin-resistant K. pneumoniae (ESC-R-Kp), and 1 carbapenemase-producing Acinetobacter baumannii simultaneously isolated from a patient transferred from Macedonia. Susceptibility tests were performed using a microdilution MIC system. The complete genome sequences were obtained by using both short-read and long-read whole-genome sequencing technologies.Results.All CPE presented high-level resistance to all aminoglycosides due to the expression of the armA 16S rRNA methylase. In C. sedlakii and E. coli (ST69), both the carbapenemase blaNDM-1 and armA genes were located on an identical IncC plasmid of type 1a. The K. pneumoniae (ST268) and P. stuartii carried chromosomal blaNDM-1 and blaOXA-48, respectively, while the ESC-R-Kp (ST395) harbored a plasmid-located blaCTX-M-15. In the latter 3 isolates, armA-harboring IncC plasmids similar to plasmids found in C. sedlakii and E. coli were also detected. The A. baumannii strain possessed the blaOXA-40 carbapenemase gene.Conclusions.The characterization of the genetic organization of IncC-type plasmids harbored by 3 different species from the same patient offered insights into the evolution of these broad-host-range plasmids. Moreover, we characterized here the first complete genome sequence of a carbapenemase-producing C. sedlakii strain, providing a reference for future studies on this rarely reported species.

Identification of Three Novel PmGRI1 Genomic Resistance Islands and One Multidrug Resistant Hybrid Structure of Tn7-like Transposon and PmGRI1 in Proteus mirabilis.

The widespread use of antibiotics in large-scale livestock production has led to serious antibiotic resistance. Proteus mirabilis is an important pathogenic bacterium on large-scale farms. Chromosomally localized mobilizable genetic elements (genomic islands) and mobile genetic elements (Tn7-like transposons) play an important role in the acquisition and transmission of resistance genes by P. mirabilis. To study the prevalence and resistance characteristics of antibiotic-resistant genomic islands in P. mirabilis of animal origin in China, we performed whole genome sequencing of P. mirabilis isolated from large-scale pig and chicken farms. Three new variants of PmGRI1 (HN31, YN8, and YN9), and a hybrid structure (HN2p) formed by the multidrug-resistant Tn7-like-HN2p transposon and a genomic island PmGRI1-HN2p, were identified from P. mirabilis. All variants underwent homologous recombination mediated by insertion sequence IS26. A genomic rearrangement in the chromosome between the Tn7-like-HN2p transposon and PmGRI1-HN2p occurred in HN2p. The heterozygous structure contained various antimicrobial resistance genes, including three copies of fluoroquinolone resistance gene qnrA1 and 16S rRNA methylase gene rmtB, which are rarely found in P. mirabilis. Our results highlight the structural genetic diversity of genomic islands by characterizing the novel variants of PmGRI1 and enrich the research base of multidrug resistance genomic islands.