Methylase Gene Research Articles

Emergence of rmtD1 gene in clinical isolates of Pseudomonas aeruginosa carrying blaKPC and/or blaVIM-2 genes in Brazil.

The aim of the present study is to describe clinical aminoglycoside- or carbapenem-resistant Pseudomonas aeruginosa isolates collected between 2018 and 2019 in a hospital in Recife City, Northeastern Brazil. It was done based on phenotypic and molecular markers of antimicrobial resistance, as well as on the clonal diversity of the investigated isolates. Thirty-four carbapenem- and/or aminoglycoside-resistant P. aeruginosa isolates were collected in a hospital in Recife City-PE, Brazil. Their antimicrobial susceptibility profile was identified based on the automated BD Phoenix ™ system. In addition, broth microdilution was performed to determine the MICs of tobramycin and polymyxin B. Eventually, isolates were subjected to PCR and sequencing in order to detect the carbapenemase enzyme (blaKPC, blaNDM, blaVIM, blaSPM-1, and blaIMP) and 16S rRNA methylase (armA, rmtB, rmtD, rmtF, and rmtG) genes; ERIC-PCR was conducted for clonal profile determination purposes. Thirty-four of the 64 isolates evaluated in the present study were selected for complementary molecular phenotypic tests, based on sample inclusion criteria. The blaKPC and blaVIM-2 genes were identified in 32.4% (11/34) and 38.2% (13/34) of tested isolates, respectively. The rmtD1 gene was detected in 32.4% (11/34) of analyzed isolates. Eight isolates carried both the blaKPC and rmtD1 genes, whereas blaVIM-2 and rmtD1 genes co-occurrence was detected in three strains; one isolate had all blaKPC, blaVIM-2, and rmtD1 genes. ERIC-PCR molecular typing has evidenced cross-transmission of three pathogenic clones among patients in the hospital. The present study is a pioneer in describing isolates harboring both blaVIM-2 and rmtD1 genes. Moreover, it emphasizes the need of conducting local molecular epidemiology studies at different time intervals in order to monitor measures adopted to prevent nosocomial infections in different hospital units.

Analysis of Relationships between Immune Checkpoint and Methylase Gene Polymorphisms and Outcomes after Unrelated Bone Marrow Transplantation.

Simple SummaryHematopoietic stem-cell transplantation (HSCT) is a curative therapy for blood disorders. Unrelated bone marrow transplantation (uBMT) is a type of allogeneic HSCT that uses the bone marrow of an unrelated donor. While HLA mismatch is a risk factor for poor outcomes in HSCT, such as graft-versus-host disease (GVHD), the importance of non-HLA single-nucleotide polymorphisms (SNPs) remains unclear. The clinical application of immune checkpoint and chromatin methylation inhibitors to cancer has been attracting attention. In the present study, we retrospectively genotyped five SNPs in four immune checkpoint genes, BTLA, PD-1, LAG3, and CTLA4, and two SNPs in methylase genes, DNMT1 and EZH2, in 999 uBMT pairs. Although no correlations were observed between these SNPs and post-uBMT outcomes, recipient EZH2 SNP exhibited a low p-value in the analysis of grade 2–4 acute GVHD (p = 0.010). This SNP may be useful for outcome predictions and needs to be confirmed in a larger-scale study.Unrelated bone marrow transplantation (uBMT) is performed to treat blood disorders, and it uses bone marrow from an unrelated donor as the transplant source. Although the importance of HLA matching in uBMT has been established, that of other genetic factors, such as single-nucleotide polymorphisms (SNPs), remains unclear. The application of immunoinhibitory receptors as anticancer drugs has recently been attracting attention. This prompted us to examine the importance of immunoinhibitory receptor SNPs in uBMT. We retrospectively genotyped five single-nucleotide polymorphisms (SNPs) in the immune checkpoint genes, BTLA, PD-1, LAG3, and CTLA4, and two SNPs in the methylase genes, DNMT1 and EZH2, in 999 uBMT donor–recipient pairs coordinated through the Japan Marrow Donor Program matched at least at HLA-A, -B, and -DRB1. No correlations were observed between these SNPs and post-uBMT outcomes (p > 0.005). This result questions the usefulness of these immune checkpoint gene polymorphisms for predicting post-BMT outcomes. However, the recipient EZH2 histone methyltransferase gene SNP, which encodes the D185H substitution, exhibited a low p-value in regression analysis of grade 2–4 acute graft-versus-host disease (p = 0.010). Due to a low minor allele frequency, this SNP warrants further investigation in a larger-scale study.

Antibiotic-resistant bacteria in municipal sewage water joining river Ganga, at Prayagraj (India)

Microbial infections are frequently occurring worldwide due to the presence of antibiotic-resistant genes in the environment. Antibiotics are becoming ineffective in the treatment of diseases due to the resistance in bacterial pathogens against these drugs. Recently, there is an incredible challenge for researchers and medical professionals in the treatment of these infectious diseases. In this study, water samples were collected from the junction of river and untreated municipal sewage water and inoculated in an antibiotic supplemented growth medium. The four isolates have been found to be ampicillin-resistant (Klebsiella pneumoniae strain AU06, Pseudomonas aeruginosa strain AU07, Aeromonas hydrophila strain AU02, and Acinetobacter baumannii strain AU01) while two isolates as erythromycin-resistant (Streptococcus pneumoniae strain AU09 and Enterococcus faecalis strain AU05). These isolates harboured beta-lactamase and ermB methylase gene conferring resistance to the bacteria. The study suggests an urgent need to stop the entry of untreated sewage water into the river to avert serious health-related issues.

Molecular survey of aminoglycoside-resistant Acinetobacter baumannii isolated from tertiary hospitals in Qazvin, Iran

Aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylases (16S RMTase) are two main resistance mechanisms against aminoglycosides. This study aimed to evaluate the frequency of AMEs and 16S rRNA methylase genes among aminoglycoside non-susceptible Acinetobacter baumannii isolates and to assess their clonal relationship using repetitive extragenic palindromic-PCR (rep-PCR). In this cross-sectional study, a total of 192 A. baumannii isolates were collected from the patients hospitalized in Qazvin, Iran (January 2016 to January 2018). Identification of isolates was performed by standard laboratory methods and API 20E strips. Antimicrobial susceptibility was determined by Kirby–Bauer method followed by examination of the genes encoding the AMEs and 16S RMTase by PCR and sequencing methods. The clonal relationship of isolates was carried out by rep-PCR. In total, 98.4% of isolates were non-susceptible to aminoglycosides, 98.4%, 97.9% and 83.9% of isolates were found to be non-susceptible against gentamicin, tobramycin and amikacin, respectively. The frequencies of aph(3′)-VI, aac(6′)-Ib, aac(3)-II, aph(3′)-Ia and armA genes were 59.3%, 39.2%, 39.2%, 31.7% and 69.8%, respectively, either alone or in combination. Rep-PCR results showed that the aminoglycoside non-susceptible isolates belonged to three distinct clones: A (79.4%), B (17.5%) and C (3.2%). The findings of this study showed a high frequency for AMEs with the emergence of armA genes among the aminoglycoside non-susceptible A. baumannii isolates. Rational administration of aminoglycosides as well as using an appropriate infection control policy may reduce the presence of resistance to antibiotics in medical centres.

Virulence Determinants and Antimicrobial Profiles of Pasteurella multocida Isolated from Cattle and Humans in Egypt.

Pasteurella multocida is a Gram-negative bacterium that causes drastic infections in cattle and humans. In this study, 55 isolates were recovered from 115 nasal swabs from apparently healthy and diseased cattle and humans in Minufiya and Qalyubia, Egypt. These isolates were confirmed by kmt1 existence, and molecular classification of the capsular types showed that types B, D, and E represented 23/55 (41.8%), 21/55 (38.1%), and 11/55 (20.0%), respectively. The isolates were screened for five virulence genes with hgbA, hgbB, and ptfA detected in 28/55 (50.9%), 30/55 (54.5%), and 25/55 (45.5%), respectively. We detected 17 capsular and virulence gene combinations with a discriminatory power (DI) of 0.9286; the most prevalent profiles were dcbF type D and dcbF type D, hgbA, hgbB, and ptfA, which represented 8/55 (14.5%) each. These strains exhibited high ranges of multiple antimicrobial resistance indices; the lowest resistances were against chloramphenicol, ciprofloxacin, amoxicillin/clavulanic acid, and levofloxacin. The macrolide–lincosamide–streptogramin B methylase gene erm(Q), with erm(42) encoding MLSB monomethyltransferase, mph(E) encoding a macrolide efflux pump, and msr(E) encoding macrolide-inactivating phosphotransferase were present. The class 1 and 2 integrons and extended-spectrum β-lactamase genes intl1, intl2, blaCTX-M, blaCTX-M-1, and blaTEM were detected. It is obvious to state that co-occurrence of resistance genes resulted in multiple drug-resistant phenotypes. The identified isolates were virulent, genetically diverse, and resistant to antimicrobials, highlighting the potential risk to livestock and humans.

A comparison of fourteen fully characterized mammalian-associated Campylobacter fetus isolates suggests that loss of defense mechanisms contribute to high genomic plasticity and subspecies evolution.

Campylobacter fetus is currently classified into three main subspecies, but only two of these, C. fetus subspecies fetus and C. fetus subsp. venerealis originate principally from ruminants where they inhabit different niches and cause distinct pathogenicity. Their importance as pathogens in international trade and reporting is also different yet the criteria defining these properties have never been fully substantiated nor understood. The situation is further compromised because the ability to differentiate between these two closely related C. fetus subspecies has traditionally been performed by phenotypic characterisation of isolates, methods which are limited in scope, time-consuming, tedious, and often yield inconsistent results, thereby leading to isolate misidentification. The development of robust genetic markers that could enable rapid discrimination between C. fetus subsp. fetus and subsp. venerealis has also been challenging due to limited differences in the gene complement of their genomes, high levels of sequence repetition, the small number of closed genome sequences available and the lack of standardisation of the discriminatory biochemical tests employed for comparative purposes. To yield a better understanding of the genomic differences that define these C. fetus strains, seven isolates were exhaustively characterised phenotypically and genetically and compared with seven previously well characterised isolates. Analysis of these 14 C. fetus samples clearly illustrated that adaption by C. fetus subsp. venerealis to the bovine reproductive tract correlated with increasing genome length and plasticity due to the acquisition and propagation of several mobile elements including prophages, transposons and plasmids harbouring virulence factors. Significant differences in the repertoire of the CRISPR (clustered regularly interspersed short palindromic repeats)-cas system of all C. fetus strains was also found. We therefore suggest that a deficiency in this adaptive immune system may have permitted the emergence of extensive genome plasticity and led to changes in host tropism through gene disruption and/or changes in gene expression. Notable differences in the sub-species complement of DNA adenine methylase genes may also have an impact. These data will facilitate future studies to better understand the precise genetic differences that underlie the phenotypic and virulence differences between these animal pathogens and may identify additional markers useful for diagnosis and sub-typing.

Emergence of 16S rRNA Methylase Gene rmtB in Salmonella Enterica Serovar London and Evolution of RmtB-Producing Plasmid Mediated by IS26.

This study aimed to characterize 16S rRNA methylase genes among Salmonella and to elucidate the structure and evolution of rmtB-carrying plasmids. One hundred fifty-eight Salmonella isolates from one pig slaughterhouse were detected as containing 16S rRNA methylase genes; two (1.27%) Salmonella London isolates from slaughtered pigs were identified to carry rmtB. They were resistant to gentamicin, amikacin, streptomycin, ampicillin, tetracycline, florfenicol, ciprofloxacin, and sulfamethoxazole/trimethoprim. The complete sequences of RmtB-producing isolates were obtained by PacBio single-molecule real-time sequencing. The isolate HA1-SP5 harbored plasmids pYUHAP5-1 and pYUHAP5-2. pYUHAP5-1 belonged to the IncFIBK plasmid and showed high similarity to multiple IncFIBK plasmids from Salmonella London in China. The rmtB-carrying plasmid pYUHAP5-2 contained a typical IncN-type backbone; the variable region comprising several resistance genes and an IncX1 plasmid segment was inserted in the resolvase gene resP and bounded by IS26. The sole plasmid in HA3-IN1 designated as pYUHAP1 was a cointegrate of plasmids from pYUHAP5-1-like and pYUHAP5-2-like, possibly mediated by IS26 via homologous recombination or conservative transposition. The structure differences between pYUHAP1 and its corresponding part of pYUHAP5-1 and pYUHAP5-2 may result from insertion, deletion, or recombination events mediated by mobile elements (IS26, ISCR1, and ISKpn43). This is the first report of rmtB in Salmonella London. IncN plasmids are efficient vectors for rmtB distribution and are capable of evolving by reorganization and cointegration. Our results further highlight the important role of mobile elements, particularly IS26, in the dissemination of resistance genes and plasmid evolution.

The Antibacterial and Antibiofilm Activity of Telithromycin Against Enterococcus spp. Isolated From Patients in China.

Telithromycin has been reported to possess robust in vitro antibacterial activity against many species of gram-positive bacteria, and telithromycin is also effective against Staphylococcus aureus biofilms. However, the in vitro antimicrobial susceptibility of telithromycin against clinical enterococci isolates in China is rarely reported and the impacts of telithromycin on the biofilm formation and eradication of enterococci remain elusive. Therefore, this study aimed to explore the inhibitory effects of telithromycin on planktonic cells and biofilms of Enterococcus strains. A total of 280 Enterococcus faecalis and 122 Enterococcus faecium isolates were collected from individual inpatients in China. The 50% minimum inhibitory concentration (MIC50) values of telithromycin against the E. faecalis and E. faecium strains carrying erythromycin-resistant methylase (erm) genes such as the ermA, ermB, or ermC, were 2 and 4 μg/mL, respectively. In addition, these isolates were typed using multilocus sequence typing (MLST) based on housekeeping genes. The predominant sequence types (STs) of E. faecalis were ST16, ST30, and ST179, and the main STs of E. faecium isolates were ST18, ST78, and ST80. Among these major STs, 87.1% (135/158) of E. faecalis and 80.4% (41/51) of E. faecium carried erm genes. Furthermore, at the subinhibitory concentrations (1/4 and 1/8 × MIC) of telithromycin, the biofilm formation of 16 E. faecalis isolates were inhibited by approximately 35%. Moreover, treatment with 8 × MIC of telithromycin or ampicillin led to an almost 40% reduction in the established biofilms of E. faecalis isolates, whereas vancomycin or linezolid with 8 × MIC had minimal effects. The combination of telithromycin and ampicillin resulted in an almost 70% reduction in the established biofilms of E. faecalis. In conclusion, these results revealed that telithromycin significantly decreased the planktonic cells of both E. faecalis and E. faecium. In addition, the data further demonstrated that telithromycin has the robust ability to inhibit E. faecalis biofilms and the combination of telithromycin and ampicillin improved antibiofilm activity. These in vitro antibacterial and antibiofilm activities suggest that telithromycin could be a potential candidate for the treatment of enterococcal infections.

N <sup>6</sup>-Adenosine Methyltransferase METTL3 is a Positive Prognostic Biomarker in Lung Squamous Cell Carcinoma

Background: N6-methyladenine is the most common modification of mRNA and IncRNA in higher organisms. m6A has been confirmed to be related to the formation and progression of tumors and m6A-related genes can be used as prognostic biomarkers in a variety of tumors. However, there have been no similar studies on lung squamous cell carcinoma. The main purpose of this study was aimed to explore the differential expression of m6A-related genes in lung squamous cell carcinoma tissues and its relationship with patient clinical prognosis. Methods: We integrated three m6A writers that catalyze the methylation of adenine on mRNA molecules. The training set including 501 patients with LUSC was collected from The Cancer Genome Atlas database and the test set including 181 patients with LUSC was collected from the Gene Expression Omnibus database. Based on the expression level of the m6A methylase gene, we established a tumor subgroup and risk-prognosis model to quantify the risk index and long-term patient prognosis, which were confirmed by principal component analysis and receiver operating characteristic curve analysis. After lung squamous cell carcinoma tissue specimens were obtained during surgery, immunohistochemistry was used to verify the results in vitro. Results: The results of the study showed that the expression of the three m6A methylases in tumor tissues and normal tissues was significantly different (P<0.05). The survival-prognostic model based on METTL3 gene expression showed better predictive performance (AUC: 0.706). Patients in the high-risk and low-risk groups exhibited significant differences in terms of survival time and 5-year and 10-year survival rates. Immunohistochemistry revealed that patients with high METTL3 expression exhibited a longer survival time than those with low METTL3 expression. Conclusions: Our study showed that the expression level of m 6 A methylase in tumor tissues was much higher than that in normal tissues. At the same time, the molecular phenotype based on the expression of METTL3 may be an independent risk factor affecting the prognosis of lung squamous cell carcinoma. These findings not only prove the important role of m6A methylase in lung squamous cell carcinoma, but are also expected to provide more accurate prognostic assessment and individualized treatment for patients with lung squamous cell carcinoma. Funding: This study was supported by the Taishan Scholar foundation (No. tshw201502061). Declaration of Interest: The authors declare that they have no conflicts of interest. Ethical Approval: The Affiliated Hospital of Qingdao University Institutional Review Board approved this study, and all study subjects provided informed consent..

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

ObjectivesCarbapenem 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. MethodsAminoglycoside 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. Results68% 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. ConclusionSuch 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.

INDUCTION OF CLINDAMYCIN RESISTANCE IN CLINICAL ISOLATES OF STAPHYLOCOCCUS AUREUS FROM A TERTIARY CARE HOSPITAL.

Nowadays in Staphylococcus aureus isolates resistant to lincosamide, macrolide and streptogramin B (MLSB) group of antibiotics are expanded. Therefore, clindamycin is preferred drug for the treatment of infections caused by S. aureus, but due to change in sensitivity patterns of clindamycin it is leading to treatment failure. The three resistance phenotypes of MLSB antibiotics are iMLSB (inducible resistance) and cMLSB (constitutive resistance) that are resistant to macrolides, lincosamides and streptogrammins B antibiotics, whereas MS resistance that is sole resistant to macrolides and streptogramins B antibiotics. Erythromycin ribosome methylase (erm) genes are responsible for expressing inducible clindamycin resistance among S. aureus. In the present investigation, a Double disc approximation/Disc induction test (D-test) and PCR were used. Out of 428 strains the prevalence of iMLSB, cMLSB and MS phenotypes were 36 (8.41%), 47 (10.98%) and 48(11.21%) respectively. It is concluded that D-test should be routinely done to avoid treatment failure due to clindamycin resistance. In addition, PCR is a simple, quick, reliable and sensitive method that could also be used in the detection of inducible clindamycin resistance. The reason for the lower prevalence of iMLSB phenotype in the present study could be due to the reason that samples included in this study were mostly from the rural areas as the exposure of antimicrobial agents is less. Keywords: Clindamycin resistance, D-test, ermA, ermC, iMLSB, S. aureus

Expression Analyses of Methylase Genes of Ppamc125 In Methylating Pbbre194 Prot-CM1 Plasmid In Escherichia Coli DH5a and TOP10

In molecular biological techniques, bacterial transformation is an essential process to strain improvement. However, transformation rate into wild type bacteria is commonly low because of the cell defense mechanism of the bacteria. Restriction modification (RM) in bacteria cells can prevent the introduction of recombinant plasmids into target bacteria. Previously, the transformation of recombinant shuttle vector pBBRE194 containing protease gene into wild type Bacillus halodurans CM1 (pBBRE194 prot-CM1) has been conducted. However, the transformation rate seemed low, and the stable recombinant clones could not be obtained. Therefore, in vivo methylation of this plasmid in E. coli has to be done before genetic transformation into the wild type bacterium, to obtain stable recombinant CM1 clones. In this study, a plasmid with artificial modification (pPAMC125) harboring genes encoding for the modification enzymes (methylases) from another strain, B. halodurans C-125, and a shuttle E.coli-Bacillus vector pBBRE194 prot-CM1 plasmid were transformed simultaneously into E. coli DH5a and TOP10, respectively. The expression of methylation enzymes genes carried by pPAMC125 in the presence of different L-arabinose concentration was observed via SDS-PAGE analyses. The analysis of the effect of these genes expression towards plasmid pBBRE194 prot-CM1 was performed by digesting the plasmid with the extracted cell of B. halodurans CM1. The digestion pattern was analyzed via DNA electrophoresis. As a result, compared to E. coli DH5a, E. coli TOP10 exhibited better condition for methylase genes expression with the result that pBBRE194 prot-CM1 plasmid was not be completely degraded by B. halodurans CM1 restriction endonuclease.

Characterization of 16S rRNA methylase genes in Enterobacterales and Pseudomonas aeruginosa in Athens Metropolitan area, 2015-2016.

The aim of this study was to characterize the 16S rRNA methylase (RMT) genes in aminoglycoside-resistant Enterobacterales and Pseudomonas aeruginosa isolates in 2015-2016 in hospitals in Athens, Greece. Single-patient, Gram-negative clinical isolates resistant to both amikacin and gentamicin (n = 292) were consecutively collected during a two-year period (2015-2016) in five tertiary care hospitals in Athens. RMT genes were detected by PCR. In all RMT-producing isolates, ESBL and carbapenemase production was confirmed by PCR, and the clonal relatedness and the plasmid contents were also characterized. None of the 138 P. aeruginosa isolates harbored any of the RMT genes surveyed although some were highly resistant to aminoglycosides (MICs > = 512 mg/L). Among 154 Enterobacterales, 31 Providencia stuartii (93.9%), 42 Klebsiella pneumoniae (37.8%), six Proteus mirabilis (75%), and two Escherichia coli (100%) isolates were confirmed as highly resistant to amikacin, gentamicin, and tobramycin with MICs ≥ 512 mg/L, harboring mainly the rmtB (98.8%). All were carbapenemase producers. P. stuartii, P. mirabilis, and E. coli produced VIM-type carbapenemases. K. pneumoniae produced KPC- (n = 34, 81.0%), OXA-48 (n = 4, 9.5%), KPC- and VIM- (n = 3, 7.1%), or only VIM-type (n = 1, 2.4%) enzymes. Two groups of similar IncC plasmids were detected one harboring rmtB1, blaVEB-1, blaOXA-10, and blaTEM-1, and the other additionally blaVIM-1 and blaSHV-5. Among RMT-producing Enterobacterales, rmtB1 predominated and was associated with carbapenemase-encoding gene(s). Similar IncC plasmids carrying a multiresistant region, including ESBL genes, and in the case of VIM-producing isolates, the blaVIM-1, were responsible for this dissemination. The co-dissemination of these genes poses a public health threat.

In vitro efficacy of ceftazidime-avibactam, aztreonam-avibactam and other rescue antibiotics against carbapenem-resistant Enterobacterales from the Arabian Peninsula

ObjectivesOur aim was to assess the susceptibility of carbapenem-resistant Enterobacterales (CRE) from the Arabian Peninsula to a broad spectrum of antibiotics, including fosfomycin, ceftazidime-avibactam, and aztreonam-avibactam. Methods1192 non-repeat CRE isolated in 2009–2017 from 33 hospitals in five countries of the Arabian Peninsula were tested. The minimum inhibitory concentration of 14 antibiotics was determined. Carbapenemase and 16S methylase genes were detected by PCR. Clonality was assessed by PFGE. ResultsThe highest rate of susceptibility was detected to aztreonam-avibactam (95.5%) followed by colistin (79.8%), fosfomycin (71.8%) and tigecycline (59.9%). Isolates co-producing two carbapenemases (12.4%) were the least susceptible. Aminoglycoside susceptibility was affected by the frequent production of a 16S methylase. Susceptibility to ceftazidime-avibactam was impacted by the high rate of metallo-beta-lactamase producers (46.3%), while aztreonam-avibactam resistance occurred mostly in clonally unrelated, carbapenemase non-producing Escherichia coli. ConclusionOf the currently available drugs: colistin, tigecycline, and ceftazidime-avibactam co-administered with aztreonam appear to be the most effective to treat CRE infections. However, the presence of non-clonal CRE isolates, in which avibactam does not lower the aztreonam MIC below the clinical breakpoint, is of notable concern. Based on the relatively high rate of fosfomycin susceptibility, it would be desirable to license parenteral fosfomycin in the region.

High Prevalence of 16s rRNA Methylase Genes Among Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae Isolates in a Chinese Tertiary Hospital.

Thirty-nine carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) isolates collected from a Chinese tertiary hospital were used in the characterization of the prevalence of 16S rRNA methylase genes. In total, 66.7% (26/39) of the CR-hvKP isolates were found to carry 16S rRNA methylase genes. The most frequently detected 16S rRNA methylase gene was armA (11/26, 42.3%), followed by rmtB (8/26, 30.8%), and coexistence of both armA and rmtB (7/26, 26.9%). All the clinical isolates were found to carry at least one carbapenemase gene, with blaKPC-2 (79.5%, 31/39), blaNDM-1 (10.3%, 4/39), and cocarrying blaKPC-2 and blaNDM-1 (10.3%, 4/39). A total of 89.7% (35/39) isolates carried extended-spectrum β-lactamase (ESBL) genes, including 61.5% (24/39) blaSHV-1, 71.8% (28/39) blaTEM-1, and 89.7% (35/39) blaCTX-M-14. All except four isolates (89.7%, 35/39) harbored quinolone resistance genes, with qnrS (82.1%, 32/39), aac(6')-Ib-cr (79.5%, 31/39), and qnrB (2.6%, 1/39). Twenty-six hvKP strains in this study were first reported to cocarry carbapenemase genes, ESBL genes, quinolone resistance genes, and 16S rRNA methylase genes simultaneously. Multilocus sequence typing (MLST) analysis assigned the 39 CR-hvKP isolates into 4 sequence types (STs), with ST11 encompassing 79.5% of the strains. Pulsed field gel electrophoresis (PFGE) typing showed that strains closely related by MLST clustered in major PFGE clusters, of which cluster A accounts for 31 ST11 isolates. Cumulatively, 16S rRNA methylase genes are highly prevalent in CR-hvKP clinical isolates especially for ST11; it is, therefore, critical to continuously monitor the epidemiology of these 16S rRNA methylase-producing CR-hvKP while simultaneously minimizing potential risks from aminoglycoside-resistant CR-hvKP.

Systematic Analysis of the DNA Methylase and Demethylase Gene Families in Rapeseed (Brassica napus L.) and Their Expression Variations After Salt and Heat stresses.

DNA methylation is a process through which methyl groups are added to the DNA molecule, thereby modifying the activity of a DNA segment without changing the sequence. Increasing evidence has shown that DNA methylation is involved in various aspects of plant growth and development via a number of key processes including genomic imprinting and repression of transposable elements. DNA methylase and demethylase are two crucial enzymes that play significant roles in dynamically maintaining genome DNA methylation status in plants. In this work, 22 DNA methylase genes and six DNA demethylase genes were identified in rapeseed (Brassica napus L.) genome. These DNA methylase and DNA demethylase genes can be classified into four (BnaCMTs, BnaMET1s, BnaDRMs and BnaDNMT2s) and three (BnaDMEs, BnaDML3s and BnaROS1s) subfamilies, respectively. Further analysis of gene structure and conserved domains showed that each sub-class is highly conserved between rapeseed and Arabidopsis. Expression analysis conducted by RNA-seq as well as qRT-PCR suggested that these DNA methylation/demethylation-related genes may be involved in the heat/salt stress responses in rapeseed. Taken together, our findings may provide valuable information for future functional characterization of these two types of epigenetic regulatory enzymes in polyploid species such as rapeseed, as well as for analyzing their evolutionary relationships within the plant kingdom.

Occurrence of NDM-1-producing Morganella morganii and Proteus mirabilis in a single patient in Portugal: probable in vivo transfer by conjugation.

To decipher the genetics of acquisition of carbapenemase-encoding genes identified in two carbapenem-resistant Enterobacteriaceae recovered from a single patient in Portugal. Carbapenemase genes were searched by PCR assays and mating-out assays were performed to further characterize the plasmid support of the carbapenemase genes. Genetic characterization of the plasmid supports was performed by whole-plasmid sequencing using the Illumina technology. We identified here two NDM-1-producing isolates, namely a Morganella morganii and a Proteus mirabilis, sharing the same blaNDM-1-positive plasmid. This 154 kb plasmid belonged to the IncA/C2 type, recently renamed IncC, and co-harboured two AmpC β-lactamase genes, namely blaCMY-4 and blaDHA-1, in addition to the 16S rRNA methylase gene armA encoding high-level resistance to aminoglycosides. In addition, the M. morganii isolate produced the CTX-M-33 extended-spectrum β-lactamase possessing weak carbapenemase activity, encoded by another plasmid. We showed here that, in addition to KPC-type and OXA-181 carbapenemases, which have been identified as widespread in this country, another concern is the emergence of NDM-1-producing enterobacterial isolates in Portugal. We demonstrated here the in vivo plasmid transfer of a blaNDM-1-positive plasmid leading to dissemination of this carbapenemase gene within different enterobacterial species in a single patient.

Assessment of 16srRNA Methylase Genes Among Non-ESBL and ESBL-Producing Klebsiella pneumoniae Isolates

: The aim of our study was to investigate mechanisms of aminoglycoside resistance in extended-spectrum beta-lactamases (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) isolates from Iran. To this end, 154 clinical isolates of K. pneumoniae were collected from two hospitals in Ilam city, Iran. The Kirby-Bauer (agar diffusion) antibiotic testing method was used to determine the susceptibility pattern of the isolates against kanamycin, gentamicin, tobramycin, netilmicin and amikacin. Aminoglycoside acetyltransferases (aac(3)-IIa, aac(6’)-Ib, and aac(3)-Ia), 16SrRNA methylase genes (armA and rmtB) and ESBL genes (blaTEM, blaSHV, and blaCTX-M) were detected by PCR amplification. 59.1% (n = 91) of K. pneumoniae isolates were detected ESBL producers with the phenotypic test. Moreover, blaTEM, blaSHV and blaCTX-M were detected in 83.5% (n=76), 52.7% (n=48) and 26.4% (n=24) of the ESBL-producing isolates, respectively. Among 52 resistant or intermediate isolates against aminoglycosides, the aac(3)-IIa, aac(6’)-Ib and rmtB genes were detected in 55.8% (n = 29), 80.8% (n = 42) and 1.9% (n = 1) of the isolates, respectively; none of the isolates, however, had the aac(3)-Ia and armA genes. Therefore, the results showed the high prevalence of aminoglycosides resistance in the K. pneumoniae isolates. As observed, the acetyltransferase modifying enzymes (aac genes) played major roles in determining this resistance. However, the rate of 16srRNA methylase genes was extremely low in K. pneumoniae.

High-Level Resistance of Toxigenic Clostridioides difficile Genotype to Macrolide-Lincosamide- Streptogramin B in Community Acquired Patients in Eastern China.

BackgroundClostridioides difficile resistant to macrolide-lincosamide-streptogramin B (MLSB) has not been reported in China.MethodsIn a cross-sectional study in two tertiary hospitals, C. difficile isolates from stool specimens from community-onset, hospital-associated diarrheal patients were analyzed for toxin genes, genotype, and antibiotic resistance, and the patients’ clinical charts were reviewed.ResultsA total of 190 (15.2%) isolates (102 A+B+ and 88 A−B+) from 1250 community acquired (CA) patients were recovered and all were susceptible to vancomycin and metronidazole. High-level resistance (minimum inhibitory concentration > 128 mg/L) to erythromycin and clindamycin was recorded in 77.9% and 88.4% of the tested isolates, respectively. Furthermore, 89.3% (159/178) of the isolates resistant to MLSB carried the erythromycin resistance methylase gene (ermB). The statistically significant factors associated with C. difficile infection (CDI) induced by A−B+ isolates with MLSB resistance included a severity score of >2 (odds ratio [95% confidence interval], 7.43 [2.31–23.87]) and platelet count (cells × 109 cells/L) < 100 [5.19 (1.58–17.04)]. The proportion of A−B+ increased with enhanced CDI severity (x2 = 21.62, P < 0.001), which was significantly higher than that of ermB-positive A+B+ in severity score of 4 (x2 = 8.61, P = 0.003). The average severity score of ermB-positive isolates was significantly higher than that of ermB-negative isolates in A−B+ (Z = −2.41, P = 0.016).ConclusionThe ermB-positive A−B+ C. difficile with MLSB resistance is described for the first time as a potential epidemic clone inducing severe CDI in CA diarrheal patients in Eastern China.

ATIM-36. TEM-GBM-001 STUDY: AUTOLOGOUS CD34+ ENRICHED HEMATOPOIETIC PROGENITOR CELLS GENETICALLY MODIFIED FOR HUMAN INTERFERON-α2 &amp; ADMINISTERED TO PATIENTS WITH GLIOBLASTOMA &amp; AN UNMETHYLATED MGMT PROMOTER

Abstract BACKGROUND Most patients with GBM &amp; an unmethylated O-6-methylguanine-DNA methylase (MGMT) gene promoter, have a poor prognosis with approximately 20% of patients surviving to 2 years. Poor prognosis is likely related to factors including a highly immunosuppressive tumor microenvironment (TME). The TME in GBM is mainly composed of tumor associated macrophages (TAMs) &amp; microglia. A subset of tumor-infiltrating macrophages characterized by expression of the angiopoietin receptor Tie2 (TEMs) have features of M2-TAMs, promote tumor angiogenesis &amp; are infrequently found in normal organs. Tie2 is significantly upregulated upon homing to tumors. Gene therapy technology has allowed TEMs to be used as carriers for the local and tumor restricted release of interferon-α (IFN). IFN has antitumor effects, inhibits angiogenesis &amp; modulates the immune system. Cell-based delivery of IFN into the TME by TEMs is expected to provide efficacy, taking advantage of pleiotropic anti-tumor effects &amp; avoiding tolerability issues associated with systemic IFN treatment. We are currently conducting a Phase I/IIa clinical study in Milan (INCB &amp; OSR) to evaluate this therapeutic approach (Temferon) in 21 patients with GBM &amp; unmethylated MGMT promoter (EudraCT Number 2018-001404-11). Eligible patients are identified immediately after first surgical resection. After screening, harvesting of HSPCs occurs followed by 6 weeks of radiotherapy. Non-myeloablative conditioning consists of BCNU &amp; thiotepa followed by administration of non-manipulated HSPCs and Temferon. RESULTS In Part A of the study, 3 cohorts of 3 patients will receive escalating doses of Temferon. After Part A, a single dose of Temferon will be studied in a further 12 patients (Part B). Criteria for study eligibility are the same for both Part A and Part B. Part A is ongoing with the first 2 patients recruited (June 2019) and Temferon is scheduled for administration. An update on the status and progress of the study will be provided.