Molecular epidemiology of multidrug-resistant Acinetobacter baumannii isolates from a hospital in Nepal.

OBJECTIVES: The emergence of multidrug-resistant (MDR) Acinetobacter baumannii has become a serious worldwide medical problem. This study was designed to clarify the genetic and epidemiological properties of MDR A. baumannii clinical isolates. METHODS: A total of 66 MDR A. baumannii isolates were obtained from 66 inpatients between May 2019 and February 2020 in a university hospital in Nepal. Whole genomes of these isolates were sequenced using next generation sequencing. Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence typing (MLST) and clonal complex (CC) analysis were conducted, and drug resistance genes were identified. RESULTS: Of the 66 isolates, 26 harbored a gene encoding NDM-type metallo-ß-lactamase, and 55 harbored a gene encoding the 16S rRNA methyltransferase, ArmA. All isolates had point mutations in the quinolone-resistance-determining regions of gyrA and parC. Phylogenetic analysis showed that 55 isolates harbored armA, 26 harbored blaNDM-1, and14 harbored blaPER-7. MLST and CC analysis revealed that 34 isolates belonged to CC2 (ST2), 10 to CC1 (nine ST1 and one ST623), and eight to CC149 (ST149). Compared to our previous study on MDR A. baumannii in Nepal in 2012, the isolation rate of CC2 increased, whereas that of CC149 decreased between 2012 and 2020. CONCLUSIONS: This study indicates that MDR A. baumannii producing carbapenemase and 16S rRNA methyltransferase, with high resistance to carbapenems and/or aminoglycosides, are spreading in medical settings in Nepal. The genetic backgrounds of MDR A. baumannii isolates have shifted to international clone 2 over several years.

Multidrug-resistant Klebsiella pneumoniae clinical isolates producing NDM- and OXA-type carbapenemase in Nepal.

OBJECTIVES: The emergence of multidrug-resistant Klebsiella pneumoniae has become a serious problem in medical settings worldwide. METHODS: A total of 46 isolates of multidrug-resistant K. pneumoniae were obtained from 2 hospitals in Nepal from October 2018 to April 2019. RESULTS: Most of these isolates were highly resistant to carbapenems, aminoglycosides, and fluoroquinolones with the minimum inhibitory concentrations (MICs) of more than 64 µg/mL. These isolates harboured carbapenemase-encoding genes, including blaNDM-1, blaNDM-5, blaOXA-181 and blaOXA-232, and 16S rRNA methyltransferase-encoding genes, including armA, rmtB, rmtC, and rmtF. Multilocus sequence typing revealed that 44 of 46 isolates were high-risk clones such as ST11 (2%), ST14 (4%), ST15 (11%), ST37 (2%), ST101 (2%), ST147 (28%), ST231 (13%), ST340 (4%), and ST395 (28%). In particular, ST395 isolates, which spread across medical settings in Nepal, co-harboured blaNDM-5 and rmtB on IncFII plasmids and co-harboured blaOXA-181/-232 and rmtF on ColKP3 plasmids. Several isolates harboured blaOXA-181 or blaNDM-5 on their chromosomes and multi-copies of blaNDM-1 or genes encoding 16S rRNA methyltransferases on their plasmids. CONCLUSIONS: The presented study demonstrates that the high-risk clones of multidrug-resistant K. pneumoniae spread in a clonal manner across hospitals in Nepal.

Global scenario of the RmtE pan-aminoglycoside-resistance mechanism: emergence of the rmtE4 gene in South America associated with a hospital-related IncL plasmid.

Antimicrobial resistance (AMR) mechanisms, especially those conferring resistance to critically important antibiotics, are a great concern for public health. 16S rRNA methyltransferases (16S-RMTases) abolish the effectiveness of most clinically used aminoglycosides, but some of them are considered sporadic, such as RmtE. The main goals of this work were the genomic analysis of bacteria producing 16S-RMTases from a 'One Health' perspective in Venezuela, and the study of the epidemiological and evolutionary scenario of RmtE variants and their related mobile genetic elements (MGEs) worldwide. A total of 21 samples were collected in 2014 from different animal and environmental sources in the Cumaná region (Venezuela). Highly aminoglycoside-resistant Enterobacteriaceae isolates were selected, identified and screened for 16S-RMTase genes. Illumina and Nanopore whole-genome sequencing data were combined to obtain hybrid assemblies and analyse their sequence type, resistome, plasmidome and pan-genome. Genomic collections of rmtE variants and their associated MGEs were generated to perform epidemiological and phylogenetic analyses. A single 16S-RMTase, the novel RmtE4, was identified in five Klebsiella isolates from wastewater samples of Cumaná. This variant possessed three amino acid modifications with respect to RmtE1-3 (Asn152Asp, Val216Ile and Lys267Ile), representing the most genetic distant among all known and novel variants described in this work, and the second most prevalent. rmtE variants were globally spread, and their geographical distribution was determined by the associated MGEs and the carrying bacterial species. Thus, rmtE4 was found to be confined to Klebsiella isolates from South America, where it was closely related to ISVsa3 and an uncommon IncL plasmid related with hospital environments. This work uncovered the global scenario of RmtE and the existence of RmtE4, which could potentially emerge from South America. Surveillance and control measures should be developed based on these findings in order to prevent the dissemination of this AMR mechanism and preserve public health worldwide.

Overexpression of 18S rRNA methyltransferase CrBUD23 enhances biomass and lutein content in Chlamydomonas reinhardtii.

Post-transcriptional modification of nucleic acids including transfer RNA (tRNA), ribosomal RNA (rRNA) and messenger RNA (mRNA) is vital for fine-tunning of mRNA translation. Methylation is one of the most widespread post-transcriptional modifications in both eukaryotes and prokaryotes. HsWBSCR22 and ScBUD23 encodes a 18S rRNA methyltransferase that positively regulates cell growth by mediating ribosome maturation in human and yeast, respectively. However, presence and function of 18S rRNA methyltransferase in green algae are still elusive. Here, through bioinformatic analysis, we identified CrBUD23 as the human WBSCR22 homolog in genome of the green algae model organism Chlamydonomas reinhardtii. CrBUD23 was a conserved putative 18S rRNA methyltransferase widely exited in algae, plants, insects and mammalians. Transcription of CrBUD23 was upregulated by high light and down-regulated by low light, indicating its role in photosynthesis and energy metabolism. To characterize its biological function, coding sequence of CrBUD23 fused with a green fluorescence protein (GFP) tag was derived by 35S promoter and stably integrated into Chlamydomonas genome by glass bead-mediated transformation. Compared to C. reinhardtii wild type CC-5325, transgenic strains overexpressing CrBUD23 resulted in accelerated cell growth, thereby leading to elevated biomass, dry weight and protein content. Moreover, overexpression of CrBUD23 increased content of photosynthetic pigments but not elicit the activation of antioxidative enzymes, suggesting CrBUD23 favors growth and proliferation in the trade-off with stress responses. Bioinformatic analysis revealed the G1177 was the putative methylation site in 18S rRNA of C. reinhardtii CC-849. G1177 was conserved in other Chlamydonomas isolates, indicating the conserved methyltransferase activity of BUD23 proteins. In addition, CrTrm122, the homolog of BUD23 interactor Trm112, was found involved in responses to high light as same as CrBUD23. Taken together, our study revealed that cell growth, protein content and lutein accumulation of Chlamydomonas were positively regulated by the 18S rRNA methyltransferase CrBUD23, which could serve as a promising candidate for microalgae genetic engineering.

Functional Genomics Identified Novel Genes Involved in Growth at Low Temperatures in Listeria monocytogenes.

Listeria monocytogenes (Lm) is a foodborne pathogen that can cause severe human illness. Standard control measures for restricting bacterial growth, such as refrigeration, are often inadequate as Lm grows well at low temperatures. To identify genes involved in growth at low temperatures, a powerful functional genomics method Tn-seq was performed in this study. This genome-wide screening comprehensively identified the known and novel genetic determinants involved in low-temperature growth. A novel gene lmo1366, encoding rRNA methyltransferase, was identified to play an essential role in Lm growth at 16°C. In contrast, the inactivation of lmo2301, a gene encoding the terminase of phage A118, significantly enhanced the growth of Lm at 16°C. The deletion of lmo1366 or lmo2301 resulted in cell morphology alterations and impaired the growth rate in milk and other conditions at low temperatures. Transcriptomic analysis revealed that the Δlmo1366 and Δlmo2301 mutants exhibited altered transcriptional patterns compared to the wild-type strain at 16°C with significant differences in genes involved in ribosome structural stability and function, and membrane biogenesis, respectively. This work uncovered novel genetic determinants involved in Lm growth at 16°C, which could lead to a better understanding of how bacteria survive and multiply at low temperatures. Furthermore, these findings could potentially contribute to developing novel antibacterial strategies under low-temperature conditions. IMPORTANCE Listeria monocytogenes is a Gram-positive pathogen that contributes to foodborne outbreaks due to its ability to survive at low temperatures. However, the genetic determinants of Lm involved in growth at low temperatures have not been fully defined. Here, the genetic determinants involved in the low-temperature growth of Lm were comprehensively identified on a genome-wide scale by Tn-seq. The gene lmo1366, encoding rRNA methyltransferase, was identified essential for growth under low-temperature conditions. On the other hand, the gene lmo2301, encoding terminase of phage A118, plays a negative role in bacterial growth at low temperatures. The transcriptomic analysis revealed the potential mechanisms. These findings lead to a better understanding of how bacteria survive and multiply at low temperatures and could provide unique targets for novel antibacterial strategies under low-temperature conditions.

High prevalence of ArmA-16S rRNA methyltransferase among aminoglycoside-resistant Klebsiella pneumoniae bloodstream isolates.

Introduction. Aminoglycosides are used for the treatment of carbapenemase-producing Klebsiella pneumoniae (CPK) infections. 16S rRNA methyltransferases (RMTs) confer resistance to all aminoglycosides and are often cocarried with NDM.Hypothesis/Gap Statement. There is a dart of studies looking at the aminoglycoside resistance mechanisms for invasive CPK isolates, particularly in OXA-48 endemic settings.Aim. We aimed to determine the prevalence of RMTs and their association with beta lactamases and MLSTs amongst aminoglycoside-resistant CPK bloodstream isolates in an OXA-48 endemic setting.Methodology. CPK isolates (n=181), collected as part of a multicentre cohort study, were tested for amikacin, gentamicin and tobramycin susceptibility using custom-made sensititre plates (GN2XF, Thermo Fisher Scientific). All isolates were previously subjected to whole-genome sequencing. Carbapenemases, RMTs, MLSTs and plasmid incompatibility groups were detected on the assembled genomes.Results. Of the 181 isolates, 109(60 %) were resistant to all three aminoglycosides, and 96 of 109(88 %) aminoglycoside-resistant isolates carried an RMT (85 ArmA, 10 RmtC, 4 RmtF1; three isolates cocarried ArmA and RmtC). Main clonal types associated with ArmA were ST2096 (49/85, 58 %) and ST14 (24/85, 28 %), harbouring mainly OXA-232 and OXA-48 +NDM, respectively. RmtC was cocarried with NDM (5/10) on ST395, and NDM +OXA-48 or NDM +KPC (4/10) on ST14, ST15 and ST16. All RMT producers also carried CTX-M-15, and the majority cocarried SHV-106, TEM-150 and multiple other antibiotic resistance genes. The majority of the isolates harboured a combination of IncFIB, IncH and IncL/M type plasmids. Non-NDM producing isolates remained susceptible to ceftazidime-avibactam.Conclusion. Aminoglycoside resistance amongst CPK bloodstream isolates is extremely common and mainly driven by clonal spread of ArmA carried on ST2096 and ST14, associated with OXA-232 and OXA48 +NDM carriage, respectively.

Mosaic Ends Tagmentation (METa) Assembly for Highly Efficient Construction of Functional Metagenomic Libraries.

Functional metagenomic libraries, physical bacterial libraries which allow the high-throughput capture and expression of microbiome genes, have been instrumental in the sequence-naive and cultivation-independent exploration of metagenomes. However, preparation of these libraries is often limited by their high DNA input requirement and their low cloning efficiency. Here, we describe a new method, mosaic ends tagmentation (METa) assembly, for highly efficient functional metagenomic library preparation. We applied tagmentation to metagenomic DNA from soil and gut microbiomes to prepare DNA inserts for high-throughput cloning into functional metagenomic libraries. The presence of mosaic end sequences in the resulting DNA fragments synergized with homology-based assembly cloning to result in a 300-fold increase in cloning efficiency compared to traditional blunt-cloning-based protocols. We show that compared to published libraries prepared by state-of-the-art protocols, METa assembly is on average ca. 20- to 200-fold more efficient and can prepare gigabase-sized libraries with as little as 200 ng of input DNA. We show the usefulness of METa assembly first by using a normative 5-µg mass of soil metagenomic DNA to prepare a 700-Gb library that allowed us to discover novel nourseothricin resistance genes and a potentially new mode of resistance to this antibiotic and second by using only 300 ng of goose fecal metagenomic DNA to prepare a 27-Gb library that captured numerous tetracycline and colistin resistance genes. METa assembly provides a streamlined, flexible, and efficient method for preparing functional metagenomic libraries, enabling new avenues of genetic and biochemical research into low-biomass or scarce microbiomes. IMPORTANCE Medically and industrially important genes can be recovered from microbial communities by high-throughput sequencing, but precise annotation is often limited to characterized genes and their relatives. Cloning a metagenome en masse into an expression host to produce a functional metagenomic library, directly connecting genes to functions, is a sequence-naive and cultivation-independent method to discover novel genes. The process of preparing these libraries is DNA greedy and inefficient, however. Here, we describe a library preparation method that is an order of magnitude more efficient and less DNA greedy. This method is consistently efficient across libraries prepared from cultures, a soil microbiome, and a goose fecal microbiome and allowed us to discover new antibiotic resistance genes and mechanisms. This library preparation method will potentially allow the functional metagenomic exploration of microbiomes that were previously off limits due to their rarity or low microbial biomass, such as biomedical swabs or exotic samples.

Vertical and horizontal dissemination of an IncC plasmid harbouring rmtB 16S rRNA methylase gene, conferring resistance to plazomicin, among invasive ST258 and ST16 KPC-producing Klebsiella pneumoniae.

OBJECTIVES: Carbapenem resistance in Klebsiella pneumoniae is a major clinical challenge. Aminoglycosides remain an important asset in the current therapeutic arsenal to treat these infections. We examined aminoglycoside resistance phenotypes and genomics in a collection of 100 invasive KPC-producing K. pneumoniae isolates sequentially collected in a Brazilian tertiary hospital between 2014 and 2016. METHODS: Aminoglycoside susceptibility testing was performed. We used a combined long-read (MinION) and short-read (Illumina) whole-genome sequencing strategy to provide a genomic picture of aminoglycoside resistance genes, with particular emphasis on 16S rRNA methyltransferases and related plasmids. RESULTS: 68% of the strains were resistant to gentamicin and 42% to amikacin, with 35% resistant to both of these commonly used aminoglycosides. We identified the 16S rRNA methyltransferase gene rmtB in 30% of these isolates: 97% (29/30) belonged to sequence type 258 (ST258) and a single isolate to the emergent ST16 clone. In ST258 and ST16 the rmtB gene was located on large IncC plasmids of 177 kb and 174 kb, respectively, highly similar to a plasmid previously identified in Proteus mirabilis in the same hospital. Moreover, 99% of the isolates remained susceptible to the veterinary-approved drug apramycin, currently under clinical development for human medicine. CONCLUSION: Such findings in geographically and temporally related isolates suggest a combination of vertical clonal spread as well as horizontal interspecies and intraspecies plasmid transfer. This broad rmtB dissemination in an endemic setting for KPC-producing clones is worrisome since it provides resistance to most clinically available aminoglycosides, including the novel aminoglycoside-modifying enzyme-resistant plazomicin.

Structural and thermodynamic characterization of a highly stable conformation of Rv2966c, a 16S rRNA methyltransferase, at low pH.

Rv2966c is a highly specific methyltransferase that methylates G966 at the N2 position in 16S rRNA of mycobacterial ribosome and can be secreted inside the host cell to methylate host DNA. However, how the secreted protein retains its structure and function in the harsh environment of host cell, remains unclear. In this work, we investigate structural features of Rv2966c at pH 4.0 and pH 7.5 by far-UV- and near-UV-circular dichroism (CD) and fluorescence spectroscopy, to gain insights into its folding and stability at the acidic pH, that it is likely to encounter inside the macrophage. We show that Rv2966c exists in a compact, folded state at both pH 7.5 and pH 4.0, a result corroborated by molecular dynamics simulations as a function of pH. In fact, Rv2966c was found to be more stable at pH 4.0 than pH 7.5, as evidenced by thermal-induced CD and nanodifferential scanning fluorimetry, and urea-induced denaturation measurements. Interestingly, unlike pH 7.5 (two-state unfolding), denaturation of Rv2966c at pH 4.0 occurs in a biphasic (N ↔ X ↔ U) manner. Further spectroscopic characterization of 'X' state, identifies characteristics of a molten globule-like intermediate. We finally conclude that Rv2966c maintains a compact folded state at pH 4.0 akin to that at pH 7.5 but with higher stability.

Comparative in vitro activity of plazomicin and older aminoglyosides against Enterobacterales isolates; prevalence of aminoglycoside modifying enzymes and 16S rRNA methyltransferases.

Comparative in vitro activity of plazomicin and 4 older aminoglycosides was evaluated with broth microdilution in 714 blood isolates from 14 hospitals in Turkey. Isolates included Escherichia coli (n=320), Klebsiella spp. (n=294), Enterobacter spp. (n=69), Serratia marcescens (n=20), and Citrobacter spp. (n=11). Isolates resistant to older aminoglycosides (n=240) were screened for aminoglycoside modifying enzyme genes: aac(6')-Ib, aac(3)-Ia, aac(3)-IIa, ant(2″)-Ia. Isolates with high MICs for plazomicin (n=41) were screened for 16S rRNA methyltransferase genes (armA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, rmtG, rmtH, npmA) and 2 carbapenemase genes (blaOXA-48, blaNDM-1). Overall, resistance to plazomicin, amikacin, netilmicin, gentamicin, and tobramycin was 7.7%, 7.4%, 31.5%, 32.9%, and 34.7%, respectively. aac(6')-Ib and aac(3)-IIa were the most common AME genes. Co-occurrence of blaNDM-1 with armA and rmtC and blaOXA-48 with armA was striking. Enterobacter cloacae carrying rmtC+blaNDM-1, S. marcescens with armA+blaOXA-48, and rmtF+ blaOXA-48 in K. pneumoniae were reported for the first time.

Systematic research to overcome newly emerged multidrug-resistant bacteria.

In the 1980s, I found that the chromosomal ß-lactamase of Klebsiella pneumoniae LEN-1 showed a very high similarity to the R-plasmid-mediated penicillinase TEM-1 on the amino acid sequence level, and this strongly suggested the origination of TEM-1 from the chromosomal penicillinases of K. pneumoniae or related bacteria. Moreover, the chromosomal K1 ß-lactamase (KOXY) of Klebsiella oxytoca was found to belong to the class A ß-lactamases that include LEN-1 and TEM-1, although KOXY can hydrolyze cefoperazone (CPZ) like the chromosomal AmpC-type cephalosporinases of various Enterobacteriaceae that can hydrolyze several cephalosporins including CPZ. Furthermore, my collaborators and I found plural novel serine-type ß-lactamases, such as MOX-1, SHV-24, TEM-91, CTX-M-64, CMY-9, CMY-19, GES-3, GES-4, and TLA-3, mediated by plasmids. Besides these serine-type ß-lactamases, we also first identified exogenously acquired metallo-ß-lactamases (MBLs), IMP-1 and SMB-1, in imipenem-resistant Serratia marcescens, and the IMP-1-producing S. marcescens TN9106 became the index case for carbapenemase-producing Enterobacteriaceae. I developed the sodium mercaptoacetic acid (SMA)-disk test for the simple identification of MBL-producing bacteria. We were also the first to identify a variety of plasmid-mediated 16S ribosomal RNA methyltransferases, RmtA, RmtB, RmtC, and NpmA, from various Gram-negative bacteria that showed very high levels of resistance to a wide range of aminoglycosides. Furthermore, we first found plasmid-mediated quinolone efflux pump (QepA) and fosfomycin-inactivating enzymes (FosA3 and FosK). We also first characterized penicillin reduced susceptible Streptococcus agalactiae, macrolide-resistant Mycoplasma pneumoniae, as well as Campylobacter jejuni, and Helicobacter pylori, together with carbapenem-resistant Haemophilus influenzae. We constructed a PCR-based open reading frame typing method for rapid identification of Acinetobacter baumannii international clones.

Genome and plasmid context of two rmtG-carrying Enterobacter hormaechei isolated from urinary tract infections in Brazil.

OBJECTIVES: Enterobacter hormaechei is an important causative agent of severe infections in critically ill patients. Aminoglycosides are among the main antibiotics for the treatment of E. hormaechei infections, however the development of antimicrobial resistance is an increasing problem. RmtG is a 16S rRNA methyltransferase, a class of enzymes conferring high-level resistance to clinically relevant aminoglycosides. The aim of this study was to characterise the full genetic context of plasmids harbouring the rmtG gene in two aminoglycoside-resistant E. hormaechei isolated in Brazil. METHODS: ThermtG-harbouring plasmids were transferred to an Escherichia coli J53 recipient strain and were fully sequenced using a MiSeq sequencing system. Complete genome assemblies were accomplished using a combination of Newbler v.3.0, SPAdes 3.10.0 and phrap/cross_match programs. Plasmid sequences were annotated using RAST server and were then manually curated using BLAST databases and ISfinder. Easyfig 2.0 was used to map and compare regions of interest containing rmtG in both plasmids. RESULTS: Both isolates carried thermtG gene on an IncA/C plasmid of ˜152kb and ˜235kb, respectively, associated with a Tn3 transposon. The plasmids contain a transfer region as well as genes involved in plasmid stability and resistance to ß-lactams, sulfonamides and quaternary ammonium compounds. One of the plasmids also carried the mrk operon encoding type 3 fimbriae. CONCLUSION: This first detection ofrmtG in E. hormaechei supports the ability for horizontal transfer. The location in complex genetic platforms carried by Tn3 transposons in IncA/C plasmids may facilitate dissemination to other Gram-negative pathogens, further limiting treatment options.

Aminoglycoside resistance determinants in multiresistant Escherichia coli and Klebsiella pneumoniae clinical isolates from Turkish and Syrian patients.

Escherichia coli and Klebsiella pneumoniae are frequently found resistance to aminoglycosides in Turkey. The aim of this study was to investigate aminoglycoside resistance in clinical isolates of E. coli and K. pneumoniae from Turkey using both phenotypic and genotypic methods and screening for the prevalence of gene coding for common aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylase genes. A total of 88 consecutive, non-duplicated E. coli (n = 65) and K. pneumoniae (n = 23) isolates showing resistance or intermediate resistance to amikacin and/or gentamicin were collected between October 2013 and May 2015 from clinical samples received at Gaziantep Dr. Ersin Arslan Training and Research Hospital. Seventeen isolates were obtained from Syrian patients. Isolates resistant to any of the two aminoglycosides were tested by PCR for seven AME genes, and 22 isolates with amikacin MIC ≥16 mg/L were also tested for 16S rRNA methylase genes. In E. coli isolates, the most frequent genes were aac(6')-Ib (50 strains; 76.9%) and aac(3)-IIa (40 strains; 70.7%), followed by aph(3')-Ia (5 strains; 7.6%) and ant(2″)-Ia (2 strains; 3.1%). Among the 23 resistant K. pneumoniae isolates, the most prevalent gene was aac(3')-IIa (87.0%) followed by aac(6')-Ib (73.9%) and aph(3')-Ia (8.6%). The rmtC gene was detected in one K. pneumoniae isolate. Resistance to aminoglycosides in clinical isolates of E. coli and K. pneumoniae from our center is predominantly caused by AAC(6')-Ib and AAC(3)-II enzymes, while the occurrence of 16S rRNA methylases is so far limited.

Frequency of 16S ribosomal RNA methyltransferase detection among Escherichia coli and Klebsiella pneumoniae clinical isolates obtained from patients in Canadian hospitals (CANWARD, 2013-2017).

Pan-aminoglycoside (amikacin, gentamicin, tobramycin, plazomicin)-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates from patients in Canadian Hospitals (2013-2017) were evaluated by whole genome sequencing for 16S ribosomal RNA methyltransferase genes. The rmtB gene was detected in 2 isolates (1 of 3094 E. coli [0.03%], 1 of 1039 K. pneumoniae [0.1%]).

Targeting the Ribosome Biogenesis Key Molecule Fibrillarin to Avoid Chemoresistance.

BACKGROUND: Inherent or acquired chemo resistance in cancer patients has been a perpetual limitation in cancer treatment. Expanding knowledge on essential cellular processes opens a new window for therapeutic targeting. Ribosome biogenesis is a process that shows potential due to its fundamental role in cell development and contribution to tumorigenesis as a result of its upregulation. Inhibiting components of ribosome biogenesis has been explored and has shown interesting results. Yet, an important key component, methyltransferase Fibrillarin (FBL), which influences both the abundance and composition of ribosomes, has not been exploited thus far. METHODS: In this literature review, we describe relevant aspects of ribosome biogenesis in cancer to emphasize the potential of FBL as a therapeutic target, in order to lower the genotoxic effects of anti-cancer treatment. RESULTS: Remarkably, the amplification of the 19q13 cytogenetic band, including the gene coding for FBL, correlated to cell viability and resistance in pancreatic cells as well as to a trend toward a shorter survival in pancreatic cancer patients. Targeting ribosome biogenesis, more specifically compared to the secondary effects of chemotherapeutics such as 5-fluorouracil or oxaliplatin, has been achieved by compound CX-5461. The cell dependent activity of this Pol I inhibitor has been reported in ovarian cancer, melanoma and leukemia models with active or mutated p53 status, presenting a promising mechanism to evade p53 resistance. CONCLUSION: Targeting critical ribosome biogenesis components in order to decrease the genotoxic activity in cancer cell looks promising. Hence, we believe that targeting key protein rRNA methyltransferase FBL shows great potential, due to its pivotal role in ribosome biogenesis, its correlation to an improved survival rate at low expression in breast cancer patients and its association with p53.

Occurrence of Acquired 16S rRNA Methyltransferase-Mediated Aminoglycoside Resistance in Clinical Isolates of Enterobacteriaceae within a Tertiary Referral Hospital of Northeast India.

The methylation of a ribosomal target leads to a high level of resistance to all clinically relevant aminoglycoside antibiotics, so early detection of these resistance determinants will help to reduce the incidence of treatment failures as well as lessen the dissemination rate. Here, we characterized different 16S rRNA methyltransferases responsible for aminoglycoside resistance and their epidemiological background in clinical isolates of Enterobacteriaceae in a tertiary referral hospital in India. All aminoglycoside-resistant isolates were screened for different 16S rRNA methyltransferases by PCR assay, and incompatibility typing of the conjugable plasmid harboring resistance genes was performed by PCR-based replicon typing. An assay for the stability and elimination of these resistance plasmids was performed. The coexistence of extended-spectrum ß-lactamases and metallo-ß-lactamases was also detected, and the heterogeneity of these isolates was determined by enterobacterial repetitive intergenic consensus PCR. The PCR assay revealed the presence of armA, rmtA, rmtB, rmtC, and rmtD in single and multiple combinations, and these were carried by a diverse group of Inc plasmids. Plasmids harboring these resistance determinants were highly stable and maintained until the 55th serial passage, but SDS treatment could easily eliminate the plasmids harboring the resistance determinants. The coexistence of blaTEM, blaPER, blaGES, and blaSHV, as well as blaVIM and blaNDM, within these isolates was also detected. Strains with different clonal patterns of aminoglycoside resistance were found to spread in this hospital setting. We observed that the 16S rRNA methyltransferase genes were encoded within different Inc plasmid types, suggesting diverse origins and sources of acquisition. Therefore, the present study is of epidemiological importance and can have a role in infection control policy in hospital settings.

Persistent spread of the rmtB 16S rRNA methyltransferase gene among Escherichia coli isolates from diseased food-producing animals in China.

A total of 963 non-duplicate Escherichia coli strains isolated from food-producing animals between 2002 and 2012 were screened for the presence of the 16S rRNA methyltransferase genes. Among the positive isolates, resistance determinants to extended spectrum ß-lactamases, plasmid-mediated quinolone resistance genes as well as floR and fosA/A3/C2 were detected using PCR analysis. These isolates were further subjected to antimicrobial susceptibility testing, molecular typing, PCR-based plasmid replicon typing and plasmid analysis. Of the 963 E. coli isolates, 173 (18.0%), 3 (0.3%) and 2 (0.2%) were rmtB-, armA- and rmtE-positive strains, respectively. All the 16S rRNA methyltransferase gene-positive isolates were multidrug resistant and over 90% of them carried one or more type of resistance gene. IncF (especially IncFII) and non-typeable plasmids played the main role in the dissemination of rmtB, followed by the IncN plasmids. Plasmids that harbored rmtB ranged in size from 20kb to 340kb EcoRI-RFLP testing of the 109 rmtB-positive plasmids from different years and different origins suggested that horizontal (among diverse animals) and vertical transfer of IncF, non-typeable and IncN-type plasmids were responsible for the spread of rmtB gene. In summary, our findings highlight that rmtB was the most prevalent 16S rRNA methyltransferase gene, which present persistent spread in food-producing animals in China and a diverse group of plasmids was responsible for rmtB dissemination.

Immunological evaluation of an rsmD-like rRNA methyltransferase from Wolbachia endosymbiont of Brugia malayi.

Wolbachia is a wonderful anti-filarial target with many of its enzymes and surface proteins (WSPs) representing potential drug targets and vaccine candidates. Here we report on the immunologic response of a drug target, rsmD-like rRNA methyltransferase from Wolbachia endosymbiont of Brugia malayi. The recombinant protein generated both humoral and cell-mediated response in BALB/c mice but compromised its immunity. The humoral response was transient and endured barely for six months in mice with or without B. Malayi challenge. In splenocytes of mice, the key humoral immunity mediating cytokine IL4 was lowered (IL4↓) while IFNγ, the major cytokine mediating cellular immunity was decreased along with upregulation of IL10 cytokine (IFNγ↓, IL10↑). The finding here indicates that the enzyme has low immunogenicity and triggers lowering of cytokine level in BALB/c mice. Interestingly the overall immune profile can be summed up with equivalent response generated by WSP or whole Wolbachia.

Fosfomycin Resistance in Escherichia coli, Pennsylvania, USA.

Fosfomycin resistance in Escherichia coli is rare in the United States. An extended-spectrum ß-lactamase-producing E. coli clinical strain identified in Pennsylvania, USA, showed high-level fosfomycin resistance caused by the fosA3 gene. The IncFII plasmid carrying this gene had a structure similar to those found in China, where fosfomycin resistance is commonly described.