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

<p>Abstract</p> <p>Background</p> <p>Recently, 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.</p> <p>Methods</p> <p>Between January 2006 and July 2008, 680 distinct <it>Escherichia coli </it>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 <it>armA </it>and <it>rmtB</it>, 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 <it>E. coli </it>isolates via 16S rRNA methylase-bearing plasmids. Homology of the isolates harboring 16S rRNA methylase genes was determined using pulse-field gel electrophoresis (PFGE).</p> <p>Results</p> <p>Among the 680 <it>E. coli </it>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 <it>rmtB </it>gene and only one harboring <it>armA</it>. 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 <it>rmtB</it>-positive donors. The plasmids of incompatibility groups IncF, IncK and IncN were detected in 34, 3 and 3 isolates, respectively. Upstream regions of the <it>armA </it>gene contained <it>IS</it>CR1 and <it>tnpU</it>, the latter a putative transposase gene,. Another putative transposase gene, <it>tnpD</it>, was located within a region downstream of <it>armA</it>. Moreover, a transposon, Tn<it>3</it>, was located upstream of the <it>rmtB</it>. Nineteen clonal patterns were obtained by PFGE, with type H representing the prevailing pattern.</p> <p>Conclusion</p> <p>A high prevalence of plasmid-mediated <it>rmtB </it>gene was found among clinical <it>E. coli </it>isolates from a Chinese teaching hospital. Both horizontal gene transfer and clonal spread were responsible for the dissemination of the <it>rmtB </it>gene.</p

Worldwide Disseminated armA Aminoglycoside Resistance Methylase Gene Is Borne by Composite Transposon Tn1548

The armA (aminoglycoside resistance methylase) gene, which confers resistance to 4,6-disubstituted deoxystreptamines and fortimicin, was initially found in Klebsiella pneumoniae BM4536 on IncL/M plasmid pIP1204 of ca. 90 kb which also encodes the extended-spectrum β-lactamase CTX-M-3. Thirty-four enterobacteria from various countries that were likely to produce a CTX-M enzyme since they were more resistant to cefotaxime than to ceftazidime were studied. The armA gene was detected in 12 clinical isolates of Citrobacter freundii, Enterobacter cloacae, Escherichia coli, K. pneumoniae, Salmonella enterica, and Shigella flexneri, in which it was always associated with bla(CTX-M-3) on an IncL/M plasmid. Conjugation, analysis of DNA sequences, PCR mapping, and plasmid conduction experiments indicated that the armA gene was part of composite transposon Tn1548 together with genes ant3"9, sul1, and dfrXII, which are responsible for resistance to streptomycin-spectinomycin, sulfonamides, and trimethoprim, respectively. The 16.6-kb genetic element was flanked by two copies of IS6 and migrated by replicative transposition. This observation accounts for the presence of armA on self-transferable plasmids of various incompatibility groups and its worldwide dissemination. It thus appears that posttranscriptional modification of 16S rRNA confers high-level resistance to all the clinically available aminoglycosides except streptomycin in gram-negative human and animal pathogens

Activity of aminoglycosides, including ACHN-490, against carbapenem-resistant Enterobacteriaceae isolates

Background The emergence of carbapenemases in Enterobacteriaceae is driving a search for therapeutic alternatives. We tested ACHN-490, a sisomicin derivative that evades all plasmid-mediated aminoglycoside-modifying enzymes, against 82 carbapenem-resistant Enterobacteriaceae isolates. Comparators included internationally and locally available aminoglycosides. Methods The isolates variously had KPC (n?=?12), SME-1 (n?=?1), IMP (n?=?13), VIM (n?=?5), NDM (n?=?17) or OXA-48 (n?=?19) carbapenemases, or had combinations of impermeability with AmpC (n?=?5) or extended-spectrum ß-lactamases (n?=?10). They included 53 Klebsiella spp., 19 Enterobacter spp., 6 Escherichia coli and 4 others; most were multiresistant. Genes were identified by PCR and sequencing; MICs were measured by CLSI agar dilution. Results ACHN-490 was active at =2 mg/L against all 65 isolates with carbapenem resistance mechanisms other than NDM enzyme, mostly with MICs of 0.12–0.5 mg/L; isepamicin was active against 63/65 at =8 mg/L. In contrast, 35% were resistant to gentamicin at 4 mg/L, 61% to tobramycin at 4 mg/L and 20% to amikacin at 16 mg/L. However, 16 of the 17 isolates with NDM-1 enzyme were resistant to ACHN-490, with MICs =64 mg/L, and these were cross-resistant to all other human-use aminoglycosides tested. Their behaviour was associated with ArmA and RmtC 16S rRNA methylases. Apramycin (a veterinary aminoglycoside) retained its full activity, with MICs of 4–8 mg/L versus strains with armA or rmtC, though resistance was seen in one Klebsiella pneumoniae with AAC(3)-IV (MIC =256 mg/L). Conclusions ACHN-490 has potent activity versus carbapenem-resistant isolates, except those also harbouring 16S rRNA methylases; isepamicin is also widely active, though less potent than ACHN-490. Evasion of 16S rRNA methylases by apramycin is noteworthy and may provide a starting point for future aminoglycoside development