Plasmid-mediated 16S rRNA Methylases Research Articles

FIRST DETECTION OF PLASMID-MEDIATED 16S RRNA METHYLASES AND AMINOGLYCOSIDE MODIFYING ENZYMES IN AN EXTENDED SPECTRUM Î’-LACTAMASES PSEUDOMONAS AERUGINOSA CLINICAL ISOLATES FROM ALGERIA: A NEW BACTERIAL SUCCESS TOWARD PAN-DRUG RESISTANCE?

Ponte Academic JournalMar 2020, Volume 76, Issue 3 FIRST DETECTION OF PLASMID-MEDIATED 16S RRNA METHYLASES AND AMINOGLYCOSIDE MODIFYING ENZYMES IN AN EXTENDED SPECTRUM Β-LACTAMASES PSEUDOMONAS AERUGINOSA CLINICAL ISOLATES FROM ALGERIA: A NEW BACTERIAL SUCCESS TOWARD PAN-DRUG RESISTANCE?Author(s): Addouda Abir ,Ammar Ayachi, Hafida Hassaine, Sana Dhaouadi, Leila Soufi, Messaoud Benmehidi, Ramzi Boubaker ElandalousiJ. Ponte - Mar 2020 - Volume 76 - Issue 3 doi: 10.21506/j.ponte.2020.3.19 Abstract:Abstract Introduction: The emergence of pan drug-resistant P. aeruginosa strains showing resistant to all antimicrobial agents is becoming a major public health concern. According to the previous hypotheses, these isolates acquired numerous drug-resistance determinants through the horizontal gene. To achieve this purpose, Our study conducted to characterize the genetic background of antibiotic resistance of Pseudomonas aeruginosa strains. Methodology: 51 no repetitive Pseudomonas aeruginosa strains were identified and investigated for genes encoding antimicrobial resistance determinants using polymerase chain reaction (PCR), besides, clonal relatedness of resistant isolates was analyzed by PCR ribotyping. Results: Various genes encoding antimicrobial resistance determinants were found in all isolates. In particular, the OXA-10Like gene in (44%) of strains, VIM-2 in (13,95%) and OprD in (4,65% ) ; molecular screening of 16S rRNA methyltransferase and aac (6 ') Ib was also found these genes in six strains, aac (3')Ia genes were detected in four strains. Ribotyping of resistant strains showed the presence of five different haplotypes. Conclusion: In this study, multiple antibiotic-resistance mechanisms contributed to the drug resistance of isolates were investigated. Thus, the genetic relationship between strains isolated from different units with the same genes resistance can explain the theory of strains dissemination and gene resistance in our hospital. Keywords: aminoglycoside-modifying enzymes; Clonal diversity; Pan drug-resistance; Pseudomonas aeruginosa; 16S rRNA methylase. Download full text:Check if you have access through your login credentials or your institution Username Password

First report of New Delhi metallo-β-lactamase-6 (NDM-6) among Klebsiella pneumoniae ST147 strains isolated from dialysis patients in Iran

There has been an alarming health-related concern about the growth of New Delhi metallo-β-lactamase. The aims of this study include the phenotypic detection of β-lactamases and molecular characterization of NDM in Klebsiella pneumoniae isolates in Tehran, Iran. A total of 120 K. pneumoniae isolates were collected from hospitalized haemodialysis patients, Tehran, Iran from March 2014 to February 2017. Antibiotic susceptibility tests were conducted using Kirby-Bauer disc diffusion and Broth Microdilution methods according to Clinical and Laboratory Standards Institute guidelines. Metallo-β-lactamase was detected using the Combined Disc Diffusion Test (CDDT), and production of carbapenemase was screened using the Modified Hodge Test. NDM-producing K. pneumoniae strains were screened for the presence of mcr-1 gene, β-lactamase genes, and 16S rRNA methylase genes by Polymerase Chain Reaction and sequencing. Molecular typing of the strains was determined using Repetitive Sequence Based-PCR and Multilocus Sequence Typing. The blaNDM-6 gene was detected in 3 (2.5%) out of 120 isolates from dialysis patients. Also, the three isolates were positive for blaCTX-M-15,blaTEM extended-spectrum β-lactamase genes, armA type plasmid-mediated 16S rRNA methylase and CMY-type plasmid-mediated AmpC β-lactamase.The isolates were identified as MLST sequence type 147 (ST147). This is the first report of blaNDM-6 in K. pneumoniae strains, isolated in Iran.

Identification and molecular characterisation of New Delhi metallo-β-lactamase-1 (NDM-1)- and NDM-6-producing Enterobacteriaceae from New Zealand hospitals

The global spread of New Delhi metallo-β-lactamase (NDM) is of significant public health concern. This study sought to determine whether blaNDM was present in Enterobacteriaceae isolates displaying resistance to carbapenems that were submitted to the National Antibiotic Reference Laboratory, Institute of Environmental Science and Research (Porirua, New Zealand) during 2009 and 2010. Isolates were tested for the presence of β-lactamase genes and 16S rRNA methylase genes by polymerase chain reaction (PCR) and sequencing. Plasmid transfer studies were undertaken on isolates found to be harbouring blaNDM. Molecular typing was performed by multilocus sequence typing (MLST). The blaNDM-1 gene was identified in four Enterobacteriaceae isolates (two Escherichia coli, one Klebsiella pneumoniae and one Proteus mirabilis) from four patients in New Zealand hospitals in 2009 and 2010. In addition, the blaNDM-6 gene, which differed from blaNDM-1 by a point mutation at position 698 (C→T), was also identified in an E. coli isolate from the same patient who harboured the blaNDM-1-positive P. mirabilis. All four patients had recently been hospitalised or received health care in India. Four of the isolates also produced a CTX-M-15 extended-spectrum β-lactamase and/or plasmid-mediated AmpC β-lactamase, and all five isolates harboured the plasmid-mediated 16S rRNA methylase rmtC gene. The E. coli types were diverse by MLST, and the K. pneumoniae isolate belonged to the internationally disseminated sequence type 11 (ST11) clone. These findings further illustrate the diversity of phenotypic and genotypic features found in association with blaNDM, in addition to documenting the international spread of this resistance mechanism, notably into a country with historically low rates of antimicrobial resistance.

Diverse prevalence of 16S rRNA methylase genes armA and rmtB amongst clinical multidrug-resistant Escherichia coli and Klebsiella pneumoniae isolates

In this study, 112 Escherichia coli and 55 Klebsiella pneumoniae isolates with a multidrug-resistant (MDR) phenotype were collected from 2007 to 2009. All isolates simultaneously exhibited resistance to cefotaxime (or ceftazidime), ciprofloxacin (or levofloxacin) and amikacin. Plasmid-mediated 16S rRNA methylases, including armA, rmtA, rmtB, rmtC, rmtD, rmtE and npmA, were detected by polymerase chain reaction (PCR) amplification. Common β-lactamase genes, including blaTEM, blaSHV, blaCTX-M, blaPER, blaVEB, blaGES and blaOXA, as well as plasmid-mediated blaAmpC and plasmid-mediated quinolone resistance (PMQR) determinants, including qnrA, qnrB, qnrS, qepA and aac(6′)-Ib-cr, were also screened. The transferable capacity of resistance plasmids was established by conjugation testing. The genetic relatedness of isolates was analysed by pulsed-field gel electrophoresis (PFGE). Only armA and rmtB genes were detected in this study. Data showed that 93.8% of MDR E. coli and 94.5% of MDR K. pneumoniae carried at least one of armA or rmtB. The armA and rmtB genes were present in 11.6% and 82.1% of MDR E. coli, respectively. In parallel, 58.2% and 40.0% of MDR K. pneumoniae were armA- and rmtB-positive, respectively. Furthermore, the qepA gene was present in 66.3% of rmtB-carrying MDR E. coli, but it was rarely detected in MDR K. pneumoniae. Approximately 71.9% of armA-positive MDR K. pneumoniae simultaneously co-carried qnrB and blaDHA. Moreover, 78.1% and 63.6%, respectively, of armA-positive and rmtB-positive MDR K. pneumoniae strains harboured qnr alleles and 53.1% and 59.1% harboured aac(6′)-Ib-cr. In addition, MDR E. coli strains exhibited a low prevalence of qnr alleles and aac(6′)-Ib-cr. PFGE analysis revealed divergent genetic relatedness, suggesting horizontal dissemination of armA and rmtB along with common β-lactamases and PMQR determinants amongst clinical MDR E. coli and K. pneumoniae isolates.

Outbreak of Salmonella enterica serotype Infantis producing ArmA 16S RNA methylase and CTX-M-15 extended-spectrum β-lactamase in a neonatology ward in Constantine, Algeria

Plasmid-mediated 16S rRNA methylases such as ArmA, which confer high levels of resistance to aminoglycosides, are increasingly reported in Enterobacteriaceae. This study investigated the molecular mechanism of β-lactam and aminoglycoside resistance in extended-spectrum β-lactamase (ESBL)-producing Salmonella enterica serotype Infantis isolated at the 53-bed neonatology ward of University Hospital Benabib in Constantine, Algeria. From September 2008 to January 2009, 200 S. enterica isolates were obtained from 138 patients (age range 8–80 months) hospitalised in the neonatology ward. Most isolates were from stool cultures, but also from two blood cultures and one gastric fluid. The isolates were multidrug-resistant and produced TEM-1 and CTX-M-15 enzymes as well as the 16S RNA methylase ArmA. The armA, bla CTX-M-15 and bla TEM-1 genes were located on the same 140-kb self-transferable plasmid belonging to the IncL/M incompatibility group. All of the S. Infantis isolates belonged to a single clone. Increased infection control measures and thorough biodecontamination of the rooms led to control of the outbreak but did not eradicate the epidemic strain. This study further illustrates the global emergence of ArmA methylase and its frequent association with bla CTX-M genes. Spread of 16S RNA methylase determinants at the same level as bla CTX-M genes in Enterobacteriaceae may seriously compromise the efficacy of aminoglycosides for treating Gram-negative infections.

Prevalence of plasmid-mediated 16S rRNA methylase genes among Proteus mirabilis isolates from a Chinese hospital

16S rRNA methylase-mediated high-level resistance to aminoglycosides has been reported recently in clinical isolates of Gram-negative bacilli from several countries. Five (2.5%, 5/198) of 198 isolates of Proteus mirabilis from a teaching hospital in Wenzhou, China, were positive for 16S rRNA methylase genes (one for armA, four forrmtB) and highly resistant to gentamicin, amikacin and tobramycin (MICs, ≥ 256 μg/ml). One of five isolates harboring 16S rRNA methyalse genes were extended-spectrum β-lactamases (ESBL) producer. The plasmids harboring 16S rRNA methylase genes from four out of five donors were transferred into the recipients,Escherichia coli J53. Among five isolates harboring armA and rmtB, the armA gene and the rmtB genes were located on the plasmids, as determined by Southern hybridization. The present study investigated the prevalence of 16S rRNA methylase genes in clinical isolates of P. mirabilis in China for the first time. Key words: Proteus mirabilis, 16S rRNA methylase, plasmid.

Resistant mechanisms of a special antibiotic resistance profile of Klebsiella pneumoniae to aminoglycoside

Objective To investigate the mechanism of a special antibiotic resistance profile of a Klebsiella pneumoniae strain from aminoglycoside resistance to gene patterns. Methods K-B diffusion test, E-test, and three dimensional test were used to investigate the antibiotic resistance profile. AME genes, 16S rRNA methylase genes and integron were confirmed by PCR, respectively. PCR products were then purified and sequenced. Results The special antibiotic resistance profiles were stable.One AME genes [Aac(3)- II ], one 16S rRNA methylase genes (armA), and class 1 integron were identified by PCR. Conclusions The special antibiotic resistance is not induced only by aminoglycoside itself. Aminoglycoside modifying enzymes and plasmid-mediated 16S rRNA methylases may be the major factors which confer aminoglycoside resistance on Klebsiella pneumoniae stains. Until recently, the mechanism causing double inhibition cycles is not clear and needs to be further investigated. Key words: Antibiotic resistance profile; Aminoglycoside; Gene patterns; 16S rRNA; Integron

High prevalence of plasmid-mediated 16S rRNA methylase gene rmtB among Escherichia coliclinical isolates from a Chinese teaching hospital

BackgroundRecently, production of 16S rRNA methylases by Gram-negative bacilli has emerged as a novel mechanism for high-level resistance to aminoglycosides by these organisms in a variety of geographic locations. Therefore, the spread of high-level aminoglycoside resistance determinants has become a great concern.MethodsBetween January 2006 and July 2008, 680 distinct Escherichia coli clinical isolates were collected from a teaching hospital in Wenzhou, China. PCR and DNA sequencing were used to identify 16S rRNA methylase and extended-spectrum β-lactamase (ESBL) genes, including armA and rmtB, and in situ hybridization was performed to determine the location of 16S rRNA methylase genes. Conjugation experiments were subsequently performed to determine whether aminoglycoside resistance was transferable from the E. coli isolates via 16S rRNA methylase-bearing plasmids. Homology of the isolates harboring 16S rRNA methylase genes was determined using pulse-field gel electrophoresis (PFGE).ResultsAmong the 680 E. coli isolates, 357 (52.5%), 346 (50.9%) and 44 (6.5%) isolates were resistant to gentamicin, tobramycin and amikacin, respectively. Thirty-seven of 44 amikacin-resistant isolates harbored 16S rRNA methylase genes, with 36 of 37 harboring the rmtB gene and only one harboring armA. The positive rates of 16S rRNA methylase genes among all isolates and amikacin-resistant isolates were 5.4% (37/680) and 84.1% (37/44), respectively. Thirty-one isolates harboring 16S rRNA methylase genes also produced ESBLs. In addition, high-level aminoglycoside resistance could be transferred by conjugation from four rmtB-positive donors. The plasmids of incompatibility groups IncF, IncK and IncN were detected in 34, 3 and 3 isolates, respectively. Upstream regions of the armA gene contained ISCR1 and tnpU, the latter a putative transposase gene,. Another putative transposase gene, tnpD, was located within a region downstream of armA. Moreover, a transposon, Tn3, was located upstream of the rmtB. Nineteen clonal patterns were obtained by PFGE, with type H representing the prevailing pattern.ConclusionA high prevalence of plasmid-mediated rmtB gene was found among clinical E. coli isolates from a Chinese teaching hospital. Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB gene.

Plasmid-Mediated 16S rRNA Methylases in Aminoglycoside-Resistant Enterobacteriaceae Isolates in Shanghai, China

High-level resistance to aminoglycosides produced by 16S rRNA methylases in Enterobacteriaceae isolates was investigated. The prevalences of armA in Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae were 0.6%, 3.0%, and 10%, respectively. rmtB was more prevalent than armA. Pulsed-field gel electrophoresis patterns indicated that armA and rmtB have spread horizontally and clonally.

Nomenclature of Plasmid-Mediated 16S rRNA Methylases Responsible for Panaminoglycoside Resistance

Production of 16S rRNA methylase has recently drawn attention as a novel aminoglycoside resistance mechanism in pathogenic gram-negative bacteria (1). It confers very-high-level resistance to all aminoglycosides that are currently available for parenteral formulation. Six distinct genes, rmtA, rmtB, rmtC, rmtD, armA, and npmA, encoding their respective enzymes have been identified in clinical and veterinary strains from various geographic areas, including East Asia, Europe, and the Americas, since 2003 (1, 10). NpmA is the only enzyme among them that methylates residue A1408, whereas the others methylate residue G1405, both within the aminoacyl site (A site) of the 16S rRNA (7, 10). All six genes are confirmed to be or are likely to locate on plasmids (3, 4, 10, 11, 12, 14). Recent findings also indicate that some of these genes are capable of crossing the barrier between glucose-fermenting and nonfermenting species. For instance, armA has been identified in both members of the family Enterobacteriaceae and in Acinetobacter baumannii (5, 13), and rmtD has been identified in Klebsiella pneumoniae and Pseudomonas aeruginosa (unpublished data). We will likely see an increasing number of reports about this resistance mechanism, including identification of genes encoding new 16S rRNA methylases. Historically, the nomenclature of genes and enzymes for many resistance mechanisms has become complicated and nonsystematic (6). An extreme example is that of aminoglycoside acetyltransferases, where new gene names are arbitrarily assigned from one of the two coexisting nomenclature systems (9). The situation is somewhat better with β-lactamases and macrolide resistance genes, due to a registry and guidelines, respectively (http://www.lahey.org/Studies/) (8). To prevent confusion over the nomenclature of 16S rRNA methylases, we would like to propose practical rules for the nomenclature of these enzymes, which shall apply to any relevant enzymes to be identified in the future. Currently, the highest and lowest identities of amino acid sequences among the G1405 16S rRNA methylases are 81.7% between RmtA and RmtB and 25.8% between ArmA and RmtD, respectively (2, 3). On the other hand, identities lower than 10% are observed between the G1405 16S rRNA methylases and the NpmA that methylates A1408 (10) (Table ​(Table).). Thus, we propose the following rules. A gene that has an amino acid identity greater than 95% with the closest known 16S rRNA methylase gene will be assigned a variant number starting from two in the order of the dates on which the sequences are deposited in the GenBank/EBML/DDBJ, e.g., rmtA2 and then rmtA3, analogous to the nomenclature of the qnr genes. A gene that has between 50 and 95% amino acid identity with the closest known 16S rRNA methylase gene will be assigned a new alphabet letter according to the closest existing gene name, e.g., rmtE, rmtF, armB, or armC, provided that the gene is shown to confer a consistent aminoglycoside resistance profile. A gene that has either an amino acid identity of less than 50% with the closest known 16S rRNA methylase gene or that is proven to methylate a new residue of 16S rRNA may be assigned a brand new gene name, like npmA, contingent upon demonstration of 16S rRNA methylation activity of the gene product and attributable resistant phenotype. Data regarding 16S rRNA methylase genes in pathogenic bacteria will be accumulated and provided at the following website http://www.nih.go.jp/niid/16s_database/index.html. TABLE 1. Identity of amino acid residues among the sequences of plasmid-mediated 16S rRNA methylases

Novel Plasmid-Mediated 16S rRNA Methylase, RmtC, Found in a Proteus mirabilis Isolate Demonstrating Extraordinary High-Level Resistance against Various Aminoglycosides

Proteus mirabilis ARS68, which demonstrated a very high level of resistance to various aminoglycosides, was isolated in 2003 from an inpatient in Japan. The aminoglycoside resistance of this strain could not be transferred to recipient strains Escherichia coli CSH-2 and E. coli HB101 by a general conjugation experiment, but E. coli DH5alpha was successfully transformed by electroporation with the plasmid of the parent strain, ARS68, and acquired an unusually high degree of resistance against aminoglycosides. Cloning and sequencing analyses revealed that the presence of a novel 16S rRNA methylase gene, designated rmtC, was responsible for resistance in strain ARS68 and its transformant. The G+C content of rmtC was 41.1%, and the deduced amino acid sequences of the newly identified 16S rRNA methylase, RmtC, shared a relatively low level of identity (< or = 29%) to other plasmid-mediated 16S rRNA methylases, RmtA, RmtB, and ArmA, which have also been identified in pathogenic gram-negative bacilli. Also, RmtC shared a low level of identity (< or = 28%) with the other 16S rRNA methylases found in aminoglycoside-producing actinomycetes. The purified histidine-tagged RmtC clearly showed methyltransferase activity against E. coli 16S rRNA in vitro. rmtC was located downstream of an ISEcp1-like element containing tnpA. Several plasmid-mediated 16S rRNA methylases have been identified in pathogenic gram-negative bacilli belonging to the family Enterobacteriaceae, and some of them are dispersing worldwide. The acceleration of aminoglycoside resistance among gram-negative bacilli by producing plasmid-mediated 16S rRNA methylases, such as RmtC, RmtB, and RmtA, may indeed become an actual clinical hazard in the near future.

Plasmid-mediated 16S rRNA methylases conferring high-level aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae isolates from two Taiwanese hospitals.

This study was conducted to investigate the occurrence of 16S rRNA methylases that confer high-level aminoglycoside resistance in Klebsiella pneumoniae and Escherichia coli isolates from two Taiwanese hospitals and the characteristics of these isolates. A total of 1624 K. pneumoniae and 2559 E. coli isolates consecutively collected over an 18 month period from a university hospital and seven E. coli and eight K. pneumoniae isolates that were resistant to amikacin from a district hospital were analysed. Two 16S rRNA methylase genes, armA and rmtB, were detected by PCR-based assays. beta-Lactamase characteristics were determined by phenotypic and genotypic methods. Overall, 28 armA-positive and seven rmtB-positive isolates were identified, and extended-spectrum beta-lactamases (ESBLs) were detected in 33 (94.3%) isolates. The prevalence rates of armA and rmtB at the university hospital were 0.9% (n=15) and 0.3% (n=5) in K. pneumoniae and 0.4% (n=10) and 0.04% (n=1) in E. coli. CTX-M-3, CTX-M-14, SHV-5-like ESBLs, and CMY-2 were detected alone or in combination in 21, 6, 11, and 2, respectively, of the 28 armA-positive isolates. CTX-M-14 was detected in six of the seven rmtB-positive isolates. Fingerprinting of conjugative plasmids revealed the dissemination of closely related plasmids containing both armA and bla(CTX-M-3). PFGE suggests that armA and rmtB spread by both horizontal transfer and clonal spread. This is the first report of the emergence of 16S rRNA methylases in Enterobacteriaceae in Taiwan. The spread of the multidrug-resistant isolates producing both ESBLs and 16S rRNA methylases may become a clinical problem.