Chapter 1: Introduction
1.5 Carbapenem Resistance
1.6.1 Aminoglycosides
1.6.1.2 Mechanisms of Resistance
Aminoglycoside resistance can occur through numerous mechanisms and these can all exist simultaneously in the same cell (Alekshun and Levy, 2007). The mechanisms include: point mutation in the 16S rRNA or ribosomal proteins (Galimand et al., 2005), methylation of the 16S rRNA (a mechanism found in most aminoglycoside producing organisms) (Galimand et al., 2005), reduced outer membrane permeability or inner membrane transport (Over et al., 2001), active pumping of drug through efflux pumps (Magnet et al., 2001), active swarming (non- specific mechanism recently seen in P. aeruginosa, which exhibited adaptive antibiotic resistance against several antibiotics) (Overhage et al., 2008), sequestration of the drug by binding to an acetyltransferase of very low activity (Magnet et al., 2003) and enzymatic hydrolysis of the drug (Vakulenko and Mobashery, 2003). Of all these mechanisms the two most important ones in Enterobacteriaceae are the production of a variety of hydrolytic enzymes and that of the production of ribosomal target- modifying 16S ribosomal methylases.
Aminoglycoside hydrolyzing enzymes have three varieties: aminoglycoside phospho-, acetyl-, and nucleotidyl-transferases transferring the respective groups to the drug and thus inactivating it (Ramirez and Tolmasky, 2010). Although highly effective, these enzymes have limited substrate specificities affecting selected aminoglycosides, only, while not affecting others.
Acquired 16S ribosomal RNA methyltransferases have long been known in M.
tuberculosis, Enterococci, S. aureus and in N. gonorrhoeae. Soon they were also described in other organisms, including Enterobacteriaceae (Galimand et al., 2003).
There are several varieties of these enzymes methylating the ribosomes, and hence making the drug-binding site unavailable for the attachment of aminoglycosides.
ArmA was first identified in a K. pneumoniae strain in a urine sample of a patient in Paris in 2000 (Galimand et al., 2003), although its nucleotide sequence was the same as a C. freundii clinical isolate found in Poland in 1996 (Gniadkowski et al., 1998).
RmtA was identified in a sputum sample of a patient in Japan in 1997 (Yokoyama et al., 2003).
Several 16S methylases, some with proven, while others with putative functions based on resistance phenotypes and amino acid sequence similarity, have been identified. They include ArmA, RmtA, RmtB (RmtB1 and RmtB2 alleles), RmtC, RmtD (RmtD1 and RmtD2 alleles), RmtE, RmtF, RmtG and RmtH (Table 5). These 16S- RMTases share low to high amino acid similarities between them (Doi et al., 2016).
Table 5: The most common 16S methylases causing aminoglycoside resistance in Enterobacteriaceae (Doi et al., 2016)
16S- RMTase
Common Species
Commonly
associated with Prevalence Distribution
ArmA
Klebsiella pneumoniae Acinetobacter baumannii
CTX-M ESBL NDM
carbapenemase OXA-23 carbapenemase
Very high in A baumannii High among NDM producers
Worldwide
RmtA Pseudomonas aeruginosa
— Low Japan, Korea
RmtB
Escherichia coli
K pneumoniae
CTX-M ESBL NDM
carbapenemase
High in China High among NDM producers
Worldwide
RmtC
K pneumoniae Proteus
mirabilis
NDM
carbapenemase
High among NDM producers
India, United Kingdom
RmtD
P aeruginosa K pneumoniae
CTX-M ESBL KPC
carbapenemase
Low South America
RmtE E coli CMY-2 AmpC Very low United States
RmtF K pneumoniae NDM
carbapenemase
High among NDM producers
India, United Kingdom RmtG
K pneumoniae CTX-M ESBL KPC
carbapenemase
Low South America
RmtH K pneumoniae CTX-M ESBL Very low Iraq
NpmA E coli — Very low Japan, Saudi
Arabia
There are several issues particularly concerning 16S methylases. On one hand, they confer resistance to practically all aminoglycosides, i.e. they do not exhibit such drug-specificity as the hydrolyzing enzymes do (Doi et al., 2016). Furthermore, as mostly being plasmid coded, some representatives are often associated with MDR, and in particular with carbapenem resistant strains.
ArmA is one of the most commonly encountered 16S-RMTases along with rmtB and is widely present in Enterobacteriaceae and A. baumannii. In Enterobacteriaceae it is commonly found in K. pneumoniae involved in healthcare
infections as well as other species involved in food borne and diarrheal illnesses, including S. flexneri and S. enterica (Golebiewski et al., 2007). Importantly, ArmA is particularly prevalent is Enterobacteriaceae producing NDM type carbapenemase.
After the discovery of NDM it became clear that many strains producing NDM or its variants were highly resistant to a variety of aminoglycosides including amikacin, tobramycin and gentamicin (Kumarasamy et al., 2010). Investigations of NDM carrying plasmids showed that blaNDM is frequently co-located with armA or other RMTase genes on the same plasmid (Rahman et al., 2014). ArmA has also been found in K. pneumoniae and other Enterobacteriaceae carrying KPC type carbapenemase gene blaKPC-2 in Italy and China (Mezzatesta et al., 2013; Luo et al., 2014). There also seems to be a reservoir of ArmA in food animals as it was recently reported in E. coli from chickens in China (Du et al., 2009).
RmtA was first identified in a P. aeruginosa strain from Japan in 1997 which showed high level of resistance to aminoglycosides (Yokoyama et al., 2003). A plasmid carrying the blaNDM-1 and rmtA was reported from a K. pneumoniae clinical strain, isolated from a patient hospitalized in India and later treated in Switzerland (Poirel et al., 2011).
RmtB is also associated with the blaNDM plasmid (Carattoli et al., 2012).
Therefore, the ongoing spread of the blaNDM carrying Enterobacteriaceae also helps the dissemination of RmtB. RmtB is more prevalent in food animals. In China, high rates of RmtB were identified in E. coli from pigs, farm workers and their environment (Chen et al., 2007; Deng et al., 2011), chicken (Du et al., 2009) and pets (Deng et al., 2011). RmtC also began to appear along with the blaNDM-1. It was found in 12 of 18 blaNDM-1 carrying E. coli isolates from UK, Pakistan and India (Mushtaq, et al., 2011).
In India a survey was carried out reporting 3.7% Enterobacteriaceae isolates had RmtC
along with blaNDM-1 (Hidalgo et al., 2013). RmtC has also been reported in K.
pneumoniae isolates in Nepal (Tada et al., 2013). These data suggest that RmtC most likely originated in the Indian subcontinent and is being incorporated into MDR/XDR Enterobacteriaceae especially those producing NDM type carbapenemases. Recently, the presence of rmtD1 or rmtD2 along with blaKPC- 2 was reported in Brazil in a K.
pneumoniae isolate (Bueno et al., 2013). The genes rmtD1 or rmtD2 and blaKPC-2 are located on separate plasmids. RmtF is particularly closely associated with NDM. The first RmtF producing strain was identified from a K. pneumoniae co-producing NDM from a patient in La Reunion Island (Galimand et al., 2012). RmtF has been found on class 1 integrons downstream of blaNDM (Mataseje et al., 2014). In a surveillance in India, 3.4% Enterobacteriaceae carried rmtF, and 59% of them along with blaNDM-1
(Hidalgo et al., 2013). Many isolates carrying rmtF have been reported from Australia, Nepal and United States of America (Tada et al., 2013; Lee et al., 2014; Sidjabat et al., 2015).