Incidence of the novel rmtF and rmtG methyltransferases in carbapenem-resistant Enterobacteriaceae from a hospital in India

Copyright © 2015 Filgona et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dear Editor, Aminoglycosides play a critically important role in antimicrobial therapy, mostly against the rapidly evolving β-lactamase-producing Enterobacteriaceae, often for their co-drug effect with β-lactams. However, the evolution of acquired 16S rRNA methyltransferases, which confer high-level and broad-spectrum aminoglycoside resistance, and their frequent co-occurrence with carbapenemases, constitute a major threat to this class of antibiotic [1]. Reported cases of aminoglycoside methyltransferase within carbapenemase-producing bacteria were relatively rare until the extensive worldwide spread of New Delhi metallo-beta-lactamase 1 (NDM-1) [2]. Recently, novel variants of methyltransferases have emerged, although their true prevalence and distribution are yet to be ascertained. In the present study, the prevalence of methyltransferase activity was determined in clinical isolates of carbapenem-resistant Enterobacteriaceae (CRE) from our center. Furthermore, incidence of the novel rmtF and rmtG methyltransferase genes within these isolates was reported. From 512 previously characterized (n = 761) clinical isolates of multidrug-resistant Enterobacteriaceae (MDRE) [3], carbapenem-non-susceptible isolates (CNSI) (defined as isolates intermediate or resistant to any of the carbapenems, namely ertapenem, imipenem, meropenem, and doripenem) were determined by the Kirby-Bauer disk susceptibility test. Further susceptibility tests to tigecycline, colistin, carbapenems, and third-generation cephalosporins; ceftazidime, ceftriaxone, and cefotaxime by minimum inhibitory concentration (MIC) breakpoints determined by the Clinical and Laboratory Standards Institute (CLSI)-referenced agar dilution method were performed on the CNSI and CRE, defined as isolates not susceptible to any of the carbapenems and resistant to all the third-generation cephalosporins [4]. Food and Drug Administration (FDA) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines for susceptibility were used for tigecycline and colistin, respectively [5]. E. coli ATCC 25922 was used as a control. The disk diffusion test revealed 62.1% (318/512) MDRE to be CNSI. Resistance rate by MIC breakpoint determination was 47. (Table 1). The most active agent of the carbapenems was doripenem, and ertapenem was the least active agent. Although tigecycline and colistin retained the best activity against the isolates, declining activity of tigecycline was evidenced by its poor performance on K. pneumoniae (42.5% resistance) (Table 1). Comparatively, in vitro activity of carbapenems, tigecycline, and colistin on the isolates significantly differed from each other (p = 0.00). While the Wilcoxon post-hoc test revealed significant difference between tigecycline and colistin (p = 0.04), …

Dear Editor, Aminoglycosides play a critically important role in antimicrobial therapy, mostly against the rapidly evolving β-lactamase-producing Enterobacteriaceae, often for their co-drug effect with β-lactams.However, the evolution of acquired 16S rRNA methyltransferases, which confer high-level and broad-spectrum aminoglycoside resistance, and their frequent co-occurrence with carbapenemases, constitute a major threat to this class of antibiotic [1].Reported cases of aminoglycoside methyltransferase within carbapenemase-producing bacteria were relatively rare until the extensive worldwide spread of New Delhi metallo-beta-lactamase 1 (NDM-1) [2].Recently, novel variants of methyltransferases have emerged, although their true prevalence and distribution are yet to be ascertained.In the present study, the prevalence of methyltransferase activity was determined in clinical isolates of carbapenem-resistant Enterobacteriaceae (CRE) from our center.Furthermore, incidence of the novel rmtF and rmtG methyltransferase genes within these isolates was reported.
From 512 previously characterized (n = 761) clinical isolates of multidrug-resistant Enterobacteriaceae (MDRE) [3], carbapenem-nonsusceptible isolates (CNSI) (defined as isolates intermediate or resistant to any of the carbapenems, namely ertapenem, imipenem, meropenem, and doripenem) were determined by the Kirby-Bauer disk susceptibility test.Further susceptibility tests to tigecycline, colistin, carbapenems, and thirdgeneration cephalosporins; ceftazidime, ceftriaxone, and cefotaxime by minimum inhibitory concentration (MIC) breakpoints determined by the Clinical and Laboratory Standards Institute (CLSI)-referenced agar dilution method were performed on the CNSI and CRE, defined as isolates not susceptible to any of the carbapenems and resistant to all the third-generation cephalosporins [4].Food and Drug Administration (FDA) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines for susceptibility were used for tigecycline and colistin, respectively [5].E. coli ATCC 25922 was used as a control.
The disk diffusion test revealed 62.1% (318/512) MDRE to be CNSI.Resistance rate by MIC breakpoint determination was 47.2%, 34.3%, 34.0, 30.2%, 19.1%, and 4.0% to ertapenem, imipenem, meropenem, doripenem, tigecycline, and colistin, respectively (Table 1).The most active agent of the carbapenems was doripenem, and ertapenem was the least active agent.Although tigecycline and colistin retained the best activity against the isolates, declining activity of tigecycline was evidenced by its poor performance on K. pneumoniae (42.5% resistance) (Table 1).Comparatively, in vitro activity of carbapenems, tigecycline, and colistin on the isolates significantly differed from each other (p = 0.00).While the Wilcoxon post-hoc test revealed significant difference between tigecycline and colistin (p = 0.04), both significantly differed from the carbapenems (Table 1).Although doripenem was the most active among the carbapenems (Table 1), its activity did not differ significantly from meropenem (p = 0.07); however, it differ significantly from imipenem (p = 0.03).On the other hand, meropenem did not differ significantly from imipenem (p = 0.03), indicating that doripenem had only marginal in vitro susceptibility advantage over imipenem and meropenem on MDRE, which suggests that therapeutic choices among these antibiotics may depend on other factors but not entirely on in vitro susceptibility advantage.The susceptibility profiles of K. pneumoniae and E. coli revealed consistently higher resistance rates among K. pneumoniae across carbapenems and tigecycline, except in colistin, where a low resistance rate of 8% (9/113) was observed (Table 1).
CRE was 55.9% (178/318) with E. coli (n = 64) and K. pneumoniae (n = 75), constituting the predominant number of isolates in this study.Polymerase chain reaction (PCR) amplification for the carbapenemase-encoding genes (bla NDM-1 and bla OXA- 48 ) was performed using primers specific for each gene [6].CRE isolates able to grow on brain-heart infusion (BHI) agar supplemented with 200 μg/mL each of gentamicin and amikacin were recognized to be positive for 16S rRNA methyltransferase activity, and PCR amplification for the novel N7 G1405 methyltransferase genes (rmtF and rmtG) was determined using specific primers [1,7,8].
bla NDM-1 has emerged globally as the most common type of carbapenemase-encoding gene, and its occurrence has surpassed that of other types of carbapenemases.bla NDM-1 and bla OXA-48 are endemic in India, and a prevalence rate ranging between 31.3% and 91.6% for bla NDM-1 in CRE isolates has been reported [9].The high bla NDM-1 (47.2%) and bla OXA-48 (43.8%) carriage rate observed in this study highlights the importance of these genes in the development of resistance in our center.In addition, an increasing trend in the occurrence of bla NDM-1 in Enterobacteriaceae isolates was observed.A study conducted in 2010 and a multicenter study conducted earlier reported a 6.7% and 2% bla NDM-1 occurrence rate from this center [10,11].In this study, an overall occurrence rate of 11.0% (n = 761 [3], bla NDM-1 = 84) (Table 2) was observed, signifying an increasing trend of NDM-1 in our center.
Methyltransferase activity was observed in 57.3% (102/178) of isolates (Table 2).Chi-square analysis revealed significant association between methyltransferase activity and bla NDM-1 carriage (p = 0.00).Incidence of the novel rmtF gene (Figure 1) among isolates positive for methyltransferase activity was 25.5% (26/102), and a high rate of rmtF positive isolates was found to harbor the bla NDM-1 gene (73.1% [19/26]) (Table 2).This finding corroborated a similar study on Indian isolates, which revealed that 24.2% of isolates expressing methyltransferase activity were positive for rmtF and 58.8% co-harbored rmtF and bla NDM-1 genes [1].The identification of this novel resistance determinant in Enterobacteriaceae from our center further confirms the assertion that the rmtF gene may have emerged some time ago and continued  to spread in association with the bla NDM-1 gene among clinical isolates unbeknownst.This is more likely, as screening for these determinants is yet to be incorporated into routine screening panels, and Enterobacteriaceae with such characteristic determinants were not targeted or resistance was viewed as being conferred by aminoglycosidesmodifying enzymes.The emergence of K. pneumoniae co-producing NDM-1 and RmtF methyltransferase was reported recently in the United States [12].Similarly, K. pneumoniae accounted for the highest RmtF production and RmtF and NDM-1 co-production in this study.The reported emergence of rmtF from the United Kingdom (UK), India, Nepal, the United States (US), and Saudi Arabia highlights the likely global dissemination of the rmtF gene among Enterobacteriaceae [1,[12][13][14].
Another novel variant, rmtG, recently reported in Brazil, shares the highest similarity with rmtD, which is confined to South America [8].However, 9.8% (10/102) isolates comprising K. pneumoniae (n = 7) and E. coli (n = 3) were positive for rmtG genotype (Figure 2 and Table 2), indicating that, unlike regionally confined rmtD, rmtG may have a worldwide dissemination.To the best of our knowledge, this is the first reported case of rmtG genotype among isolates of Enterobacteriaceae outside South America.Thus, more studies are needed to ascertain worldwide dissemination of this resistance determinant.The emergence and simultaneous identification of these novel methyltransferases in Asia, the UK, and the US is a reminder that it is critical not only to detect new acquired resistance determinants, but also to analyze our environment for known traits in order to gain the most knowledge [2].
Although a major limitation of this work is our inability to determine other carbapenemase-encoding genes besides bla NDM-1 and bla OXA-48 and other 16S rRNA methyltransferases besides the novel variants, the incidence of rmtF and rmtG genes in CRE from our center stresses the need to constantly monitor clinical isolates for emerging resistance patterns.As aminoglycoside and carbapenem activity becomes increasingly compromised, and because tigecycline and colistin are not ideal drugs for the treatment of infection due to MDRE, prompt identification and tracking of possible sources of infection and implementation of standard infection control practices are crucial.