armA Gene Research Articles

Prevalence of 16S rRNA Methylation Enzyme Gene armA in Salmonella From Outpatients and Food.

Salmonella is the primary cause of community-acquired foodborne infections, so its resistance to antimicrobials, such as aminoglycosides, is a public health issue. Of concern, aminoglycoside resistance in Salmonella is increasing rapidly. Here, we performed a retrospective study evaluating the prevalence of Salmonella harboring armA-mediated aminoglycoside resistance in community-acquired infections and in food or environmental sources. The prevalence rates of armA-harboring Salmonella strains were 1.1/1,000 (13/12,095) and 8.7/1,000 (32/3,687) in outpatient and food/environmental isolates, respectively. All the armA-harboring Salmonella strains were resistant to multiple drugs, including fluoroquinolone and/or extended-spectrum cephalosporins, and most (34/45) belonged to serovar Indiana. The armA gene of these strains were all carried on plasmids, which spanned five replicon types with IncHI2 being the dominant plasmid type. All the armA-carrying plasmids were transferable into Escherichia coli and Acinetobacter baumannii recipients. The conjugation experiment results revealed that the armA-harboring S. Indiana strains had a relatively higher ability to acquire armA-carrying plasmids. The low similarity of their pulsed field gel electrophoresis patterns indicates that the armA-harboring Salmonella strains were unlikely to have originated from a single epidemic clone, suggesting broad armA spread. Furthermore, the genetic backgrounds of armA-harboring Salmonella strains isolated from outpatients exhibited higher similarity to those isolated from poultry than to those isolated from swine, suggesting that poultry consumption maybe an infection source. These findings highlight an urgent need to monitor the prevalence and transmission of armA-harboring Salmonella, especially S. Indiana, to better understand the potential public health threat and prevent the further spread of these strains.

Successful treatment of infective endocarditis due to pandrug-resistant Klebsiella pneumoniae with ceftazidime-avibactam and aztreonam

Pandrug-resistant (PDR) K. pneumoniae refractory to conventional treatment has been reported worldwide, causing a huge burden on the healthcare system, patient safety and the economy. K. pneumoniae is a prominent opportunistic pathogen causing hospital-acquired and community-acquired infections, but is rarely associated with infective endocarditis. Currently, there are sparse data guiding the optimal regimen when commonly used antibiotics fail, notably for the treatment of endocarditis infections. Here we report our experience in treating a 40-year-old female with PDR K. pneumoniae infection of cardiovascular implantable electronic device (CIED) and right-sided infective endocarditis. Initial susceptibility testing of the incriminated pathogen showed an apparent susceptibility to colistin but the prolonged course of colistin, gentamicin and meropenem did not resolve the infection. However, the synergistic combinations of aztreonam with ceftazidime-avibactam was able to overcome resistance and clear the infection rapidly. Genome sequencing showed that the PDR K. pneumoniae isolate belongs to the international high-risk clone ST14. The isolate harbored genes encoding NDM-1, OXA-48, CTX-M-14b, SHV-28 and OXA-1, explaining resistance to all β-lactams, including carbapenems. It carried the armA gene conferring resistance to all clinically important aminoglycosides and had alterations in GyrA, ParC and MgrB, explaining resistance to ciprofloxacin and colistin.

Prevalence and genotypic characterization of Salmonella spp. from chicken meats marketed in the province of Skikda, Algeria.

Here, we aim to determine the prevalence of Salmonella contamination of poultry meat from butcheries of the province of Skikda and to investigate antibiotic resistance. Salmonella spp. isolates were screened from 70 samples, including chicken breasts (n = 40 samples) and chicken thighs (n = 30 samples) collected from 14 butcheries. All suspected Salmonella colonies from selective media were confirmed by MALDI-TOF MS and serotyped. The susceptibility profile to 16 antibiotics was studied. According to the antibiotic susceptibility results, resistance genes were investigated by standard PCR targeting various genes such as blaSHV, blaTEM, aac3, aac6-Ibcr, aad, qnrA and qnrB. Of the 14 butcheries studied, samples from eight butcheries were contaminated with Salmonella (57.14%). 19 Salmonella strains were isolated, including five serotypes with a predominance of Kentucky serotype (n = 9), Enteridis (n = 3), followed by Heidelberg (n = 3), Virchow (n = 3), and Manhattan (n= 1). All isolates were resistant to Rifampicin (100%; n = 19), and to other antibiotics such as Ciprofloxacin (47.36%), Amoxicillin-clavulanic acid (47.36%; n = 9), Amoxicillin, (47.36%; n = 9), Ticarcillin-clavulanic acid (47.36%; n = 9), and Gentamycin (47.36%; n = 9). All isolates showing multidrug resistance (47.36%; n = 9) were positive by PCR to the blaTEM-1 β-lactamase gene, from which 8 strains carried the aminoglycoside resistance aad7 gene. However, none was positive for the tested blaSHV, Aac3, Aac6-Ibcr, qnrA, qnrB, ArmA and ArmB genes. Our findings show a worrying rate of Salmonella contamination of poultry meats.

Molecular survey of aminoglycoside-resistant Acinetobacter baumannii isolated from tertiary hospitals in Qazvin, Iran

Aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylases (16S RMTase) are two main resistance mechanisms against aminoglycosides. This study aimed to evaluate the frequency of AMEs and 16S rRNA methylase genes among aminoglycoside non-susceptible Acinetobacter baumannii isolates and to assess their clonal relationship using repetitive extragenic palindromic-PCR (rep-PCR). In this cross-sectional study, a total of 192 A. baumannii isolates were collected from the patients hospitalized in Qazvin, Iran (January 2016 to January 2018). Identification of isolates was performed by standard laboratory methods and API 20E strips. Antimicrobial susceptibility was determined by Kirby–Bauer method followed by examination of the genes encoding the AMEs and 16S RMTase by PCR and sequencing methods. The clonal relationship of isolates was carried out by rep-PCR. In total, 98.4% of isolates were non-susceptible to aminoglycosides, 98.4%, 97.9% and 83.9% of isolates were found to be non-susceptible against gentamicin, tobramycin and amikacin, respectively. The frequencies of aph(3′)-VI, aac(6′)-Ib, aac(3)-II, aph(3′)-Ia and armA genes were 59.3%, 39.2%, 39.2%, 31.7% and 69.8%, respectively, either alone or in combination. Rep-PCR results showed that the aminoglycoside non-susceptible isolates belonged to three distinct clones: A (79.4%), B (17.5%) and C (3.2%). The findings of this study showed a high frequency for AMEs with the emergence of armA genes among the aminoglycoside non-susceptible A. baumannii isolates. Rational administration of aminoglycosides as well as using an appropriate infection control policy may reduce the presence of resistance to antibiotics in medical centres.

High-Level Aminoglycoside Resistance in Human Clinical Klebsiella pneumoniae Complex Isolates and Characteristics of armA-Carrying IncHI5 Plasmids.

Aminoglycosides are important options for treating life-threatening infections. However, high levels of aminoglycoside resistance (HLAR) among Klebsiella pneumoniae isolates have been observed to be increasing frequently. In this study, a total of 292 isolates of the K. pneumoniae complex from a teaching hospital in China were analyzed. Among these isolates, the percentage of HLAR strains was 13.7% (40/292), and 15 aminoglycoside resistance genes were identified among the HLAR strains, with rmtB being the most dominant resistance gene (70%, 28/40). We also described an armA-carrying Klebsiella variicola strain KP2757 that exhibited a high-level resistance to all aminoglycosides tested. Whole-genome sequencing of KP2757 demonstrated that the strain contained one chromosome and three plasmids, with all the aminoglycoside resistance genes (including two copies of armA and six AME genes) being located on a conjugative plasmid, p2757-346, belonging to type IncHI5. Comparative genomic analysis of eight IncHI5 plasmids showed that six of them carried two copies of the intact armA gene in the complete or truncated Tn1548 transposon. To the best of our knowledge, for the first time, we observed that two copies of armA together with six AME genes coexisted on the same plasmid in a strain of K. variicola with HLAR. Comparative genomic analysis of eight armA-carrying IncHI5 plasmids isolated from humans and sediment was performed, suggesting the potential for dissemination of these plasmids among bacteria from different sources. These results demonstrated the necessity of monitoring the prevalence of IncHI5 plasmids to restrict their worldwide dissemination.

Phenotypic and genotypic characterization of antibiotic resistance in military hospital-associated bacteria from war injuries in the Eastern Ukraine conflict between 2014 and 2020

Infections from the recent conflict in Ukraine have been poorly investigated. To describe the phenotypic and genotypic mechanisms of antibiotic resistance in pathogens associated with war injuries in the Ukraine conflict. This report describes a retrospective multi-centre microbiological survey conducted in four Ukrainian military hospitals between 2014 and 2020. The phenotypes of 813 organisms obtained from 1061 tests of 162 patients were analysed. Fifty-two isolates underwent whole-genome sequencing. Resistance was highest in Acinetobacter baumannii, with 92.5% ((48/52) 95% confidence interval (CI) 81.8-97.9) resistant to fluoroquinolones, 83.0% ((43/52) 95% CI 70.2-91.9) resistant to aminoglycosides, and 67.9% ((37/52) 95% CI 53.7-80.1) resistant to carbapenems. In contrast, resistance to carbapenems was 55.6% ((30/52) 95% CI 41.4-69.1) in Pseudomonas aeruginosa, 42.9% in Escherichia coli ((12/28) 95% CI 24.5-62.8), and 32.8% in Klebsiella pneumoniae ((20/34) 95% CI 21.3-46.0). Multi-drug-resistant strains harboured an abundance of antibiotic resistance genes. K.pneumoniae co-produced class A and D β-lactamases, in one case with blaNDM-1 and rmtC 16S rRNA methyltransferase. A.baumannii carried class A and D β-lactamases but not metallo-β-lactamases; in four isolates, carbapenemases were present with the RmtASE gene armA. P.aeruginosa harboured a wide range of class A and D β-lactamases along with metallo-β-lactamases, as well as the RmtB4 RmtASE gene. Gram-positive cocci were generally sensitive to the tested antibiotics. The incidence of resistance among the studied pathogens was higher than that in Ukrainian civilian hospitals and European countries. The discovery of P.aeruginosa, A. baumannii, and K.pneumoniae co-producing carbapenemases and RmtASEs is of particular importance, and hospitals should be vigilant for their emergence.

Prevalence of Genes Encoding Aminoglycoside-Modifying Enzymes and armA among Acinetobacter baumannii Clinical Isolates in Alexandria, Egypt

Acinetobacter baumannii (A.baumannii ) is a ubiquitous pathogen responsible for serious infections in hospitalized patients with a high propensity to develop resistance to antimicrobial agents. The study aimed to determine the antimicrobial resistance patterns and the prevalence of aminoglycoside resistance genes among A. baumannii clinical isolates from patients in different intensive care units (ICUs) in Alexandria, Egypt. A total of 100 A. baumannii isolates collected from ICU patients were confirmed as A. baumannii by VITEK 2 and the presence of the blaOXA-51 gene has been reported. Antimicrobial susceptibility testing was performed and Multiplex PCR was done for the detection of aminoglycoside resistance genes. Most of the isolates (82%) were resistant to all tested aminoglycosides; resistance was higher for kanamycin and neomycin, followed by amikacin. The predominant AMEs were aphA6 and aphA1 in 86% and 67% of the isolates, respectively; aacA4 and aacC1 were detected in 37% and 8%, respectively, while aadA1 and aadB were present in 34% and 4%, respectively. Furthermore, armA gene was detected in 83% of the isolates. The results of this study revealed a high level carriage of armA and AMEs which limit the usage of aminoglycoside as a treatment option for A. baumannii and makes treatment extremely difficult.

Detection of diverse carbapenem and multidrug resistance genes and high-risk strain types among carbapenem non-susceptible clinical isolates of target gram-negative bacteria in Kenya.

Carbapenem-resistant gram-negative bacteria are an increasingly significant clinical threat globally. This risk may be underestimated in Kenya as only four carbapenemase genes in three bacterial species have been described. The study aimed to understand the antibiotic resistance profiles, genes, sequence types, and distribution of carbapenem-resistant gram-negative bacteria from patients in six hospitals across five Kenyan counties by bacterial culture, antibiotic susceptibility testing, and whole-genome sequence analysis. Forty-eight, non-duplicate, carbapenem non-susceptible, clinical isolates were identified across the five counties (predominantly in Nairobi and Kisii): twenty-seven Acinetobacter baumannii, fourteen Pseudomonas aeruginosa, three Escherichia coli, two Enterobacter cloacae, and two Klebsiella pneumoniae. All isolates were non-susceptible to β-lactam drugs with variable susceptibility to tigecycline (66%), minocycline (52.9%), tetracycline (29.4%), and levofloxacin (22.9%). Thirteen P. aeruginosa isolates were resistant to all antibiotics tested. Eleven carbapenemase genes were identified: blaNDM-1, blaOXA-23, -58, -66, -69, and -91 in A. baumannii (STs 1, 2, 164 and a novel ST1475), blaNDM-1 in E. cloacae (STs 25,182), blaNDM-1, blaVIM-1and -6, blaOXA-50 in P. aeruginosa (STs 316, 357, 654, and1203), blaOXA-181, blaNDM-1 in K. pneumoniae (STs 147 and 219), and blaNDM-5 in E. coli (ST164). Five A. baumannii isolates had two carbapenemases, blaNDM-1, and either blaOXA-23 (4) or blaOXA-58 (1). AmpC genes were detected in A. baumannii (blaADC-25), E. cloacae (blaDHA-1 and blaACT-6, 16), and K. pneumoniae (blaCMY). Significant multiple-drug resistant genes were the pan-aminoglycoside resistance16srRNA methyltransferase armA, rmtB, rmtC, and rmtF genes. This study is the first to report blaOXA-420, -58, -181, VIM-6, and blaNDM-5 in Kenyan isolates. High-risk STs of A. baumannii (ST1475, ST2), E. cloacae ST182, K. pneumoniae ST147, P. aeruginosa (ST357, 654), and E. coli ST167, ST648 were identified which present considerable therapeutic danger. The study recommends urgent carbapenem use regulation and containment of high-risk carbapenem-resistant bacteria.

Distribution of genes encoding 16S rRNA methyltransferase in plazomicin-nonsusceptible carbapenemase-producing Enterobacterales in Brazil

Background: The presence of 16S rRNA methyltranferases (16S-RMTases) in carbapenemase-producing Enterobacterales (CPE) is a major concern because it inactivates all clinical use of aminoglycosides, including plazomicin. The aim of this study is to investigate the prevalence of 16S-RMTases in CPE nonsusceptible to plazomicin collected in different Brazilian hospitals. Methods: All isolates with plazomicin MIC ≥ 4 µg/mL (n = 67) were screened for the presence of 16S-RMTases by sequencing. Results: 54 (80.6%) isolates encoded 16S-RMTase genes (41 rmtB1, 7 armA, 3 rmtD2, 1 rmtD1 and 2 rmtC). Among 41 samples rmtB1 positive, 40 co-harbored blaKPC-2 and 1 blaOXA-48 gene. Of the seven isolates harboring armA gene, 6 were New Delhi Metallo-beta-lactamase (NDM)-producer. rmtD was only found in isolates Klebsiella pneumoniae Carbapenemase (KPC)-producers, one in Serratia marcescens with rmtD2, not reported in Brazil. Conclusion: The co-existence of 16S-RMTase and CPE is worrisome because of limited treatment options and the endemic characteristic of (KPC) and NDM in Brazil.

Acquisition of a genomic resistance island (AbGRI5) from global clone 2 through homologous recombination in a clinical Acinetobacter baumannii isolate.

To reconstruct the evolutionary history of the clinical Acinetobacter baumannii XH1056, which lacks the Oxford scheme allele gdhB. Susceptibility testing was performed using broth microdilution and agar dilution. The whole-genome sequence of XH1056 was determined using the Illumina and Oxford Nanopore platforms. MLST was performed using the Pasteur scheme and the Oxford scheme. Antibiotic resistance genes were identified using ABRicate. XH1056 was resistant to all antibiotics tested, apart from colistin, tigecycline and eravacycline. MLST using the Pasteur scheme assigned XH1056 to ST256. However, XH1056 could not be typed with the Oxford MLST scheme as gdhB is not present. Comparative analyses revealed that XH1056 contains a 52 933 bp region acquired from a global clone 2 (GC2) isolate, but is otherwise closely related to the ST23 A. baumannii XH858. The acquired region in XH1056 also contains a 34 932 bp resistance island that resembles AbGRI3 and contains the armA, msrE-mphE, sul1, blaPER-1, aadA1, cmlA1, aadA2, blaCARB-2 and ere(B) resistance genes. Comparison of the XH1056 chromosome to that of GC2 isolate XH859 revealed that the island in XH1056 is in the same chromosomal region as that in XH859. As this island is not in the standard AbGRI3 position, it was named AbGRI5. XH1056 is a hybrid isolate generated by the acquisition of a chromosomal segment from a GC2 isolate that contains a resistance island in a new location-AbGRI5. As well as generating ST256, it appears likely that a single recombination event is also responsible for the acquisition of AbGRI5 and its associated antibiotic resistance genes.

Expansion of acquired 16S rRNA methytransferases along with CTX-M-15, NDM and OXA-48 within three sequence types of Escherichia coli from northeast India

BackgroundThis study aimed to identify ten different 16S rRNA methyltransferase genes (rmtA, rmtB, rmtC, rmtD, armA, rmtF, npmA, rmtH, rmtE and rmtG) and their coexisting ESBL and carbapenemase with the emergence of three E.coli clones within a single study centre.MethodsA total of 329 non-duplicate E.coli isolates were studied to detect the presence of 16S rRNA methyltransferases along with β-lactamases (TEM, SHV, OXA, VEB, GES, PER,CTX-M types, NDM, OXA-48,VIM, IMP and KPC) using PCR assay. Horizontal transferability were validated by transformation and conjugation analysis. Plasmid incompatibility typing and MLST analysis was also performed.ResultsA total of 117 isolates were found to be resistant to at least one of the aminoglycoside antibiotics. It was observed that 77 (65.8%) were positive for 16S rRNA methyltransferases. Among them thirty nine isolates were found to harbour only blaCTX-M-15, whereas combination of genes were observed in three isolates (blaVEB+ blaCTX-M-15 in 2 isolates and blaPER + blaCTX-M-15 in 1 isolate). blaNDM and blaOXA-48 like genes were found in 23 and 9 isolates, respectively. All the resistance genes were conjugatively transferable, and incompatibility typing showed multiple 16S rRNA methyltransferase genes were originated from a single Inc. I1 group. MLST analysis detected 3 clones of E.coliST4410, ST1341 and ST3906.ConclusionThe present study identified emergence of three clones of E.coli, resistant to aminoglycoside -cephalosporin- carbapenem. This warrants immediate measures to trace their transmission dynamics in order to slow down their spread in clinical setting.

<p><em>Acinetobacter baumannii</em> Sequence Types Harboring Genes Encoding Aminoglycoside Modifying Enzymes and 16SrRNA Methylase; a Multicenter Study from Pakistan</p>

IntroductionThe aminoglycosides are widely used for the therapeutic management of infections caused by gram-negative bacteria, including the Acinetobacter baumannii strains. However, the resistance to the members of the aminoglycoside family, such as amikacin, gentamicin, and tobramycin, is increasingly being common among the clinical isolates.PurposeThis study aimed to investigate the presence of 16SrRNA methylases and aminoglycoside modifying enzymes (AMEs) genes among aminoglycoside resistant A. baumannii isolates and to study the genetic diversity of the clinical population of A. baumannii in local hospitals.Material and MethodsThe 143 A. baumannii clinical strains were analyzed for antimicrobial susceptibility, genetic screening for enzymes conferring aminoglycosides resistance followed by the multilocus sequence typing.ResultsThe 133/143 (93%) isolates were non-susceptible to at least one of the tested aminoglycosides, including amikacin, gentamicin, and tobramycin. The MIC distribution has shown that 87.486.7% strains were resistant to amikacin and gentamicin, respectively. The aphA6, aadB, aacC1, and aphA1 were found in 74.1%, 59.4%, 16.1%, and 11.2% isolates, respectively, whereas the armA was found in 28% of the strains having a higher MIC value (MIC; ≥256µg/mL). The MLST data have shown that the ST589 and ST2 were the most common STs and corresponded to 51 (35.7%) and 38 (26.6%) isolates, respectively, and few of the isolates corresponding to these STs were found to harbor the armA gene with a variable genotypic profile for AMEs.DiscussionThe study has reported the incidence of various enzymes conferring aminoglycoside resistance among the A. baumannii clones for the first time from Pakistan. The findings suggest the possibility of transmission of aminoglycoside resistance determinants through the lateral gene transfer as well as clonal dissemination.

Extensive antimicrobial resistance mobilization via multicopy plasmid encapsidation mediated by temperate phages.

ObjectivesTo investigate the relevance of multicopy plasmids in antimicrobial resistance and assess their mobilization mediated by phage particlesMethodsSeveral databases with complete sequences of plasmids and annotated genes were analysed. The 16S methyltransferase gene armA conferring high-level aminoglycoside resistance was used as a marker in eight different plasmids, from different incompatibility groups, and with differing sizes and plasmid copy numbers. All plasmids were transformed into Escherichia coli bearing one of four different lysogenic phages. Upon induction, encapsidation of armA in phage particles was evaluated using qRT–PCR and Southern blotting.ResultsMulticopy plasmids carry a vast set of emerging clinically important antimicrobial resistance genes. However, 60% of these plasmids do not bear mobility (MOB) genes. When carried on these multicopy plasmids, mobilization of a marker gene armA into phage capsids was up to 10000 times more frequent than when it was encoded by a large plasmid with a low copy number.ConclusionsMulticopy plasmids and phages, two major mobile genetic elements (MGE) in bacteria, represent a novel high-efficiency transmission route of antimicrobial resistance genes that deserves further investigation.

Abundance of Colistin-Resistant, OXA-23- and ArmA-Producing Acinetobacter baumannii Belonging to International Clone 2 in Greece.

Carbapenem resistant Acinetobacter baumannii (CRAB) represents one of the most challenging pathogens in clinical settings. Colistin is routinely used for treatment of infections by this pathogen, but increasing colistin resistance has been reported. We obtained 122 CRAB isolates from nine Greek hospitals between 2015 and 2017, and those colistin resistant (ColR; N = 40, 32.8%) were whole genome sequenced, also by including two colistin susceptible (ColS) isolates for comparison. All ColR isolates were characterized by a previously described mutation, PmrBA226V, which was associated with low-level colistin resistance. Some isolates were characterized by additional mutations in PmrB (E140V or L178F) or PmrA (K172I or D10N), first described here, and higher colistin minimum inhibitory concentrations (MICs), up to 64 mg/L. Mass spectrometry analysis of lipid A showed the presence of a phosphoethanolamine (pEtN) moiety on lipid A, likely resulting from the PmrA/B-induced pmrC overexpression. Interestingly, also the two ColS isolates had the same lipid A modification, suggesting that not all lipid A modifications lead to colistin resistance or that other factors could contribute to the resistance phenotype. Most of the isolates (N = 37, 92.5%) belonged to the globally distributed international clone (IC) 2 and comprised four different sequence types (STs) as defined by using the Oxford scheme (ST 425, 208, 451, and 436). Three isolates belonged to IC1 and ST1567. All the genomes harbored an intrinsic blaOXA–51 group carbapenemase gene, where blaOXA–66 and blaOXA–69 were associated with IC2 and IC1, respectively. Carbapenem resistance was due to the most commonly reported acquired carbapenemase gene blaOXA–23, with ISAba1 located upstream of the gene and likely increasing its expression. The armA gene, associated with high-level resistance to aminoglycosides, was detected in 87.5% of isolates. Collectively, these results revealed a convergent evolution of different clonal lineages toward the same colistin resistance mechanism, thus limiting the effective therapeutic options for the treatment of CRAB infections.

Occurrence of NDM-1-producing Morganella morganii and Proteus mirabilis in a single patient in Portugal: probable in vivo transfer by conjugation.

To decipher the genetics of acquisition of carbapenemase-encoding genes identified in two carbapenem-resistant Enterobacteriaceae recovered from a single patient in Portugal. Carbapenemase genes were searched by PCR assays and mating-out assays were performed to further characterize the plasmid support of the carbapenemase genes. Genetic characterization of the plasmid supports was performed by whole-plasmid sequencing using the Illumina technology. We identified here two NDM-1-producing isolates, namely a Morganella morganii and a Proteus mirabilis, sharing the same blaNDM-1-positive plasmid. This 154 kb plasmid belonged to the IncA/C2 type, recently renamed IncC, and co-harboured two AmpC β-lactamase genes, namely blaCMY-4 and blaDHA-1, in addition to the 16S rRNA methylase gene armA encoding high-level resistance to aminoglycosides. In addition, the M. morganii isolate produced the CTX-M-33 extended-spectrum β-lactamase possessing weak carbapenemase activity, encoded by another plasmid. We showed here that, in addition to KPC-type and OXA-181 carbapenemases, which have been identified as widespread in this country, another concern is the emergence of NDM-1-producing enterobacterial isolates in Portugal. We demonstrated here the in vivo plasmid transfer of a blaNDM-1-positive plasmid leading to dissemination of this carbapenemase gene within different enterobacterial species in a single patient.

Assessment of 16srRNA Methylase Genes Among Non-ESBL and ESBL-Producing Klebsiella pneumoniae Isolates

: The aim of our study was to investigate mechanisms of aminoglycoside resistance in extended-spectrum beta-lactamases (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) isolates from Iran. To this end, 154 clinical isolates of K. pneumoniae were collected from two hospitals in Ilam city, Iran. The Kirby-Bauer (agar diffusion) antibiotic testing method was used to determine the susceptibility pattern of the isolates against kanamycin, gentamicin, tobramycin, netilmicin and amikacin. Aminoglycoside acetyltransferases (aac(3)-IIa, aac(6’)-Ib, and aac(3)-Ia), 16SrRNA methylase genes (armA and rmtB) and ESBL genes (blaTEM, blaSHV, and blaCTX-M) were detected by PCR amplification. 59.1% (n = 91) of K. pneumoniae isolates were detected ESBL producers with the phenotypic test. Moreover, blaTEM, blaSHV and blaCTX-M were detected in 83.5% (n=76), 52.7% (n=48) and 26.4% (n=24) of the ESBL-producing isolates, respectively. Among 52 resistant or intermediate isolates against aminoglycosides, the aac(3)-IIa, aac(6’)-Ib and rmtB genes were detected in 55.8% (n = 29), 80.8% (n = 42) and 1.9% (n = 1) of the isolates, respectively; none of the isolates, however, had the aac(3)-Ia and armA genes. Therefore, the results showed the high prevalence of aminoglycosides resistance in the K. pneumoniae isolates. As observed, the acetyltransferase modifying enzymes (aac genes) played major roles in determining this resistance. However, the rate of 16srRNA methylase genes was extremely low in K. pneumoniae.

Co-expression of 16S rRNA Methyltransferase and Carbapenemase in Multidrug Resistant Gram Negative Bacteria

Introduction: The drug resistance caused by bacteria has complicated the fight against the infectious diseases and recovery of the patients from the diseases. Multiple mode of drug resistant mechanisms including epigenetic and enzymatic mode have been involved in the genesis of drug resistance leaving only fewer antibiotics for the treatment. Studies has shown the presence of 16S methyltransferase which is associated with carbapenem drug resistance. Early detection of different multimode of bacterial drug resistance helps in reducing the dissemination rate and treatment failures. Aim: To detect the genes related to the methylation mode of bacterial drug resistant mechanism including armA (aminoglycoside resistance methylase), rmtA, rmtB, rmtC and rmtD (rmt-RNA methyltrasferase) as well as genes OXA-48 (Oxacillinase), VIM (Verona intergron metallo-β lactamase) and KPC (Klebsiella pneumoniae carbapenemase) in the production of carbapenemases and also to identify the co-expression of these genes in the gram negative bacterial isolates. Materials and Methods: Prospective study was conducted through application of multiplex Polymerase Chain Reaction (PCR)for detection of 16S methyltransferase genes including armA, rmtA, rmtB, rmtC and rmtD and carbpenemase VIM, KPC and OXA-48 genes, respectively. A total of 200 multidrug resistant gram negative bacilli were evaluated for presence of these genes. The antibiotic sensitivity was evaluated using Kirby-Bauer disc diffusion method. Results: The gram negative bacteria which were isolated includes Acinetobacter spp. 20, E.coli-85, Klebsiella spp.-55 and Pseudomonas aeruginosa-40 from several specimens such as urine, endotracheal tube-secretion, wound swab, sputum, pus etc. A total of 121 (61%) of the bacterial isolates were resistant to either gentamicin or amikacin and netilimycin and 50 (25%) bacterial isolates were resistant to either imipenem or carbapenem. A total of 48 (24%) isolates were resistant to both aminoglycosides and carbapenems. The 16S methyltransferase genes including armA gene was detected in 16 isolates, rmtB in 15 isolates, rmtC in 10 isolates and rmtD detected in 13 isolates The carbapenem genes detected including VIM in 20 isolates, OXA-48 in 25 isolates and KPC was detected in 13 bacterial isolates. Co-expression of both methyltransferase and carbapenemase were detected in nine isolates. Conclusion: The presence of multiple mode of bacterial drug resistance mechanisms including epigenetic and enzymatic modes have been found to be associated with resistance to different classes of antibiotics. The application of multiplex PCR helps in detection of multiple genes involved in bacterial drug resistance and helps in prevention of rapid spread of bacterial drug resistance and prevention of overuse of antibiotics.

Clonal Dissemination of Clinical Isolates of Acinetobacter baumannii Carriers of 16S rRNA Methylase Genes in an Oncological Hospital in Recife, Brazil.

16S rRNA methylases confer high-level resistance to aminoglycosides which are used to treat serious infections caused by gram-negative bacteria, such as Acinetobacter spp. Some genes encoding these enzymes are disseminated worldwide, while others were detected in only some countries. The objective was to characterize the susceptibility profile to aminoglycosides (amikacin and gentamicin) of clinical isolates of Acinetobacter spp. from an oncological hospital in Recife, and given the resistance to both antimicrobials, to characterize minimal inhibitory concentrations (MICs) of amikacin, gentamicin and tobramycin, the occurrence of 16S rRNA methylase genes (armA, rmtB, rmtC and rmtD) and of ß-lactamase gene (blaKPC) and the clonal profile. Isolates resistant to both antimicrobials, amikacin and gentamicin, were selected by disk diffusion technique in Mueller-Hinton agar and identified. Broth microdilution was conducted to determine MICs of amikacin, gentamicin, and tobramycin. These isolates were subjected to polymerase chain reaction and pulsed-field gel electrophoresis. Among 23 analyzed isolates, 12 (52.2%) were resistant to gentamicin and amikacin and identified as Acinetobacter baumannii. Among these, 11 (91.7%), 12 (100%), and 9 (75%) isolates showed respectively MICs >256 µg/mL of amikacin, >64 µg/mL of gentamicin, and >64 µg/mL of tobramycin. The armA gene was found in 12 (100%) isolates and 6 (50%) showed coexistence of armA, rmtB, and rmtC genes. The rmtD and blaKPC genes were not detected. These isolates showed high genetic similarity (92%) and were classified as clone A. Elaboration and fulfillment of measures are thus essential to prevent the spread of this resistance mechanism.

Co occurrence of two 16S rRNA methyltrasferases along with NDM and OXA 48 like carbapenamases on a single plasmid in Klebsiella pneumoniae.

:BACKGROUND:The carbapenemase-encoding genes, blaNDM- and blaOXA-48-like, confer resistance to all the known beta-lactams and are encountered along with other beta-lactamase-encoding genes and/or 16S ribosomal RNA (rRNA)-methylating genes. The co-occurrence of blaNDM and blaOXA-48-like on a single plasmid is a rare occurrence.AIM AND OBJECTIVE:The purpose of the study was to characterize the plasmids in Klebsiella pneumoniae isolates producing 16S rRNA methyltransferase along with blaNDM, blaOXA-48-like, and other resistance encoding genes.MATERIALS AND METHODS:One-hundred and seventeen K. pneumoniae clinical isolates which were resistant to aminoglycosides were collected. Polymerase chain reaction-based screening for 16S rRNA methyltransferase genes armA, rmtB, and rmtC; carbapenamase genes blaNDM, blaOXA-48-like, blaIMP, blaVIM, and blaKPC; and other resistance genes such as blaTEM, blaSHV, blaCTX-M, and qnr (A, B, and S) determinants acc (6’) Ib-cr was performed. Conjugation experiment was carried out for seven isolates that anchored blaNDM and blaOXA-48-like along with any one of the 16S rRNA methyltransferases. The plasmid-based replicon typing for different plasmid-incompatible (Inc) group was performed on the conjugatively transferable plasmids.RESULTS:Among the 16S rRNA methyltransferases, armA was more predominant. blaNDM and blaOXA-48-like were present in 56 (47.86%) and 22 (18.80%) isolates, respectively. Out of seven isolates which were conjugatively transferable, only four had blaNDM and blaOXA-48-like on the same plasmid and they belonged to Inc N and A/C replicon. Three isolates co-harbored 16S rRNA methyltransferases armA, rmtB, and rmtC, and out of the them, one isolate harbored two 16S rRNA methyltransferases armA and rmtB, on the single-plasmid replicon A/C.CONCLUSION:This is the first report revealing the coexistence of blaNDM and blaOXA-48-like co-harboring two 16S rRNA methylases on a single conjugative plasmid replicon belonging to incompatibility group A/C.

High-Level Resistance to Aminoglycosides due to 16S rRNA Methylation in Enterobacteriaceae Isolates.

Introduction: High-level aminoglycoside resistance due to methylase genes has been reported in several countries. The purpose of this study was to investigate the diversity of the genes encoding 16S rRNA methylase and their association with resistance phenotype in Enterobacteriacae isolates. Materials and Methods: Based on sampling size formula, from February to August 2014, a total of 307 clinical Enterobacteriaceae isolates were collected from five hospitals in northwest Iran. The disk diffusion method for amikacin, gentamicin, tobramycin, kanamycin, and streptomycin, as well as the minimum inhibitory concentration (MIC) for aminoglycosides (except streptomycin), was used. Six 16S rRNA methylase genes (armA, npmA, and rmtA-D) were screened by PCR and sequencing assays. Results: In this study, 220 (71.7%) of 307 isolates were aminoglycoside resistant and 40 isolates (18.2%, 40/220) were positive for methylase genes. The frequency of armA, rmtC, npmA, rmtB, and rmtA genes was 9.5%, 4.5%, 3.6%, 2.3%, and 1%, respectively. The rmtD gene was not detected in the tested bacteria. Sixty percent of positive methylase gene isolates displayed high-level resistance (MIC ≥512 μg/mL to amikacin and kanamycin; and MIC ≥128 μg/mL to gentamicin and tobramycin). Conclusions: The prevalence of resistance to aminoglycoside in Iran is high. Furthermore, there is a statistically significant association between amikacin and kanamycin resistance with the presence of rmtC and rmtB genes.