Bleomycin Resistance Gene Research Articles

First report of coexistence of blaKPC-2 and blaNDM-1 in carbapenem-resistant clinical isolates of Klebsiella aerogenes in Brazil.

Klebsiella aerogenes is an important opportunistic pathogen with the potential to develop resistance against last-line antibiotics, such as carbapenems, limiting the treatment options. Here, we investigated the antibiotic resistance profiles of 10 K. aerogenes strains isolated from patient samples in the intensive-care unit of a Brazilian tertiary hospital using conventional PCR and a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the blaKPC-2 and blaNDM-1 genes simultaneously. All isolates were completely resistant to β-lactam antibiotics, including ertapenem, imipenem, and meropenem with differencing levels of resistance to aminoglycosides, quinolones, and tigecycline also observed. Half of the strains studied were classified as multidrug-resistant. The carbapenemase-producing isolates carried many genes of interest including: β-lactams (blaNDM-1, blaKPC-2, blaTEM-1, blaCTX-M-1 group, blaOXA-1 group and blaSHVvariants in 20-80% of the strains), aminoglycoside resistance genes [aac(6')-Ib and aph(3')-VI, 70 and 80%], a fluoroquinolone resistance gene (qnrS, 80%), a sulfonamide resistance gene (sul-2, 80%) and a multidrug efflux system transporter (mdtK, 70%) while all strains carried the efflux pumps Acr (subunit A) and tolC. Moreover, we performed a comprehensive genomic characterization of a specific K. aerogenes strain (CRK317) carrying both the blaKPC-2 and blaNDM-1 genes simultaneously. The draft genome assembly of the CRK317 had a total length of 5,462,831 bp and a GC content of 54.8%. The chromosome was found to contain many essential genes. In silico analysis identified many genes associated with resistance phenotypes, including β-lactamases (blaOXA-9, blaTEM-1, blaNDM-1, blaCTX-M-15, blaAmpC-1, blaAmpC-2), the bleomycin resistance gene (bleMBL), an erythromycin resistance methylase (ermC), aminoglycoside-modifying enzymes [aac(6')-Ib, aadA/ant(3")-Ia, aph(3')-VI], a sulfonamide resistance enzyme (sul-2), a chloramphenicol acetyltransferase (catA-like), a plasmid-mediated quinolone resistance protein (qnrS1), a glutathione transferase (fosA), PEtN transferases (eptA, eptB) and a glycosyltransferase (arnT). We also detected 22 genomic islands, eight families of insertion sequences, two putative integrative and conjugative elements with a type IV secretion system, and eight prophage regions. This suggests the significant involvement of these genetic structures in the dissemination of antibiotic resistance. The results of our study show that the emergence of carbapenemase-producing K. aerogenes, co-harboring blaKPC-2 and blaNDM-1, is a worrying phenomenon which highlights the importance of developing strategies to detect, prevent, and control the spread of these microorganisms.

Molecular Characterization of pBOq-IncQ and pBOq-95LK Plasmids of Escherichia coli BOq 01, a New Isolated Strain from Poultry Farming, Involved in Antibiotic Resistance.

The increase in antimicrobial resistance has raised questions about how to use these drugs safely, especially in veterinary medicine, animal nutrition, and agriculture. Escherichia coli is an important human and animal pathogen that frequently contains plasmids carrying antibiotic resistance genes. Extra chromosomal elements are required for various functions or conditions in microorganisms. Several phage-like plasmids have been identified, which are important in antibiotic resistance. In this work, the molecular characterization of the pBOq-IncQ (4.5 kb) and pBOq-95LK (95 kb) plasmids found in the E. coli strain BOq 01, a multidrug resistant bacteria isolated from a poultry farm, are considered. Plasmid pBOq-IncQ belongs to the incQ incompatibility plasmid family and is involved in sulfonamide resistance. Plasmid pBOq-95LK is a lytic phage-like plasmid that is involved in the lysis of the E. coli BOq 01 strain and carries a bleomycin resistance gene and a strain cured of this plasmid shows bleomycin sensitivity. Induction of the lytic cycle indicates that this phage-like plasmid is an active phage. This type of plasmid has been reported to acquire genes such as mcr-1, which codes for colistin resistance and bacterial persistence and is a significant public health threat. A genome comparison, a pangenomic and phylogenomic analysis with other phage-like plasmids reported in the literature were performed to understand better the evolution of this kind of plasmid in bacteria and its potential importance in antibiotic resistance.

A Longitudinal Survey of Antibiotic-Resistant Enterobacterales in the Irish Environment, 2019–2020

The natural environment represents a complex reservoir of antibiotic-resistant bacteria as a consequence of different wastewater discharges including anthropogenic and agricultural. Therefore, the aim of this study was to examine sewage and waters across Ireland for the presence of antibiotic-resistant Enterobacterales. Samples were collected from the West, East and South of Ireland. Two periods of sampling took place between July 2019 and November 2020, during which 118 water (30 L) and 36 sewage samples (200 mL) were collected. Waters were filtered using the CapE method, followed by enrichment and culturing. Sewage samples were directly cultured on selective agars. Isolates were identified by MALDI-TOF and antibiotic susceptibility testing was performed in accordance with EUCAST criteria. Selected isolates were examined for blaCTX-M, blaVIM, blaIMP, blaOXA-48, blaNDM, and blaKPC by real time PCR and whole genome sequencing (n = 146). A total of 419 Enterobacterales (348 water, 71 sewage) were isolated from all samples. Hospital sewage isolates displayed the highest percentage resistance to many beta-lactam and aminoglycoside antibiotics. Extended-spectrum beta-lactamase-producers were identified in 78% of water and 50% of sewage samples. One or more carbapenemase-producing Enterobacterales were identified at 23 individual sampling sites (18 water, 5 sewage). This included the detection of blaOXA-48 (n = 18), blaNDM (n = 14), blaKPC (n = 4) and blaOXA-484 (n = 1). All NDM-producing isolates harbored the ble-MBL bleomycin resistance gene. Commonly detected sequence types included Klebsiella ST323, ST17, and ST405 as well as E. coli ST131, ST38 and ST10. Core genome MLST comparisons detected identical E. coli isolates from wastewater treatment plant (WWTP) influent and nursing home sewage, and the surrounding waters. Similarly, one Klebsiella pneumoniae isolated from WWTP influent and the surrounding estuarine water were identical. These results highlight the need for regular monitoring of the aquatic environment for the presence of antibiotic-resistant organisms to adequately inform public health policies.

Whole Genome Sequence Analysis of Multidrug Resistant Escherichia coli and Klebsiella pneumoniae Strains in Kuwait.

The spread of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae is a global concern. The management of infections caused by multidrug resistance (MDR) isolates poses substantial clinical challenges in both hospitals and communities. This study aimed to investigate the genetic characteristics and variations of MDR E. coli and K. pneumoniae isolates. Bacterial identification and antibiotic susceptibility testing against 19 antibiotics were performed by standard methods. Whole genome sequencing (WGS) was carried out on eight carbapenem-resistant isolates using an Illumina MiSeq platform. The assembled draft genomes were annotated, then sequences were blasted against antimicrobial resistance (AMR) genes database. WGS detected several resistance genes mediating the production of β-lactamases, including carbapenems and extended-spectrum β-lactamase genes as (blaOXA-1/-48, blaKPC-2/-29, blaCMY-4/-6, blaSHV-11/-12, blaTEM-1, blaCTX-M-15, blaOKP-B, blaACT and blaEC). Furthermore quinolone resistance including oqxA/oqxB, aac(6′)-Ib-cr5, gyrA_D87N, gyrA_S83F, gyrA_S83L, parC_S80I, parE_S458A, parE_I355T, parC_S80I, and qnrB1. In addition to aminoglycoside modifying enzymes genes (aph(6)-Id, aph(3″)-Ib, aac(3)-IIa, aac(6′)-Ib, aadA1, aadA2 and aadA5), trimethoprim-sulfamethoxazole (dfrA12/A14/A17 and sul1/sul2), tetracycline (tetA and tetB), fosfomycin (fosA and uhpT_E350Q) resistance genes, while other genes were detected conferring chloramphenicol (floR, catA2, and efflux pump cmIA5), macrolides resistance (mph(A) and erm(B), and quaternary ammonium efflux pump qacEdelta. Bleomycin and colistin resistance genes were detected as ble and pmrB_R256G, respectively. Comprehensive analysis of MDR strains provided by WGS detected variable antimicrobial resistance genes and their precise resistance mechanism. WGS is essential for control and prevention strategies to combat the growing threat of AMR and the implementation of multifaceted interventions are needed.

Genomic characterisation of multidrug-resistant Bacillus toyonensis strain 4HC1 isolated from marine plastic in Norway

Bacillus toyonensis is widespread in nature. Multidrug-resistant B. toyonensis strain 4HC1 was isolated from polyethylene submerged in the water column near a beach in Øygarden, Norway. We analysed the whole genome sequence of strain 4HC1 in order to understand the genetic basis of the observed phenotypic antibiotic resistance. Whole-genome sequencing of B. toyonensis strain 4HC1 was performed on Illumina MiSeq platform using 2×300 bp chemistry. The genome sequence was assembled using SPAdes v.3.13.0 and was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The draft genome of strain 4HC1 is 6 156 259 bp (133 contigs) in size with a GC content of 34.95%. The genome comprises 6089 protein-coding genes, 86 tRNAs and 24 rRNAs. Strain 4HC1 is resistant to cefotaxime, trimethoprim and ampicillin and carries various antibiotic resistance genes (ARGs), including several β-lactamases, aminoglycoside 6-adenylyltransferase, a TetM family tetracycline resistance gene, two different tetracycline efflux pumps, and a bleomycin resistance gene. Several virulence genes including genes involved in immune evasion, iron acquisition and toxins were also detected in strain 4HC1. The draft genome sequence of B. toyonensis strain 4HC1 released here shows the presence of various ARGs and virulence genes in a multidrug-resistant strain isolated from marine plastic.

Intron-mediated enhancement of transgene expression in the oleaginous diatom Fistulifera solaris towards bisabolene production

Microalgae have been widely recognized as promising hosts for production of various valuable compounds. However, low expression or silencing of transgenes in most microalgae species have hindered their broader implementation in biotechnological applications. In many eukaryotes, intron mediated enhancement (IME) of gene expression by inclusion of introns in heterologous sequences has resulted in a significant increase in transgene expression. This phenomenon has been found in a broad range of eukaryotic organisms including animals, plants, yeasts, and more recently green microalgae. However, no investigation has been conducted in diatoms. Here, we present findings of IME in the oleaginous diatom, Fistulifera solaris. Inclusion of a native intron found in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene into a bleomycin-resistance gene (shble) resulted in enhancement of the mRNA level of shble, leading to increase in resistance and selection. We tested this IME in a heterologous plant sesquiterpene synthase gene construct (AgBS), leading to expression approximately double that of non-intron containing cassettes. The heterologous AgBS lead to accumulation of the sesquiterpene (E)-α-bisabolene, indicating that native precursors were amenable to manipulation and available in the cytoplasm of this diatom. To the best of our knowledge, this is the first study to investigate the effect of IME in diatoms.

Generation of two ISL1-tdTomato reporter human induced pluripotent stem cell lines using CRISPR-Cas9 genome editing

ISL1 encodes a member of the LIM/homeodomain family of transcription factors. This encoded protein plays central roles in the development of motor neuron, pancreas, and secondary heart field. Here we generated heterozygous fluorescent reporters of the ISL1 gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock-in 2A-tdTomato and EF1 alpha promoter-driven Bleomycin resistance gene to the translational ISL1 C-terminal region. The resulting ISL1-TEZ lines showed tdTomato fluorescence upon motor neuron differentiation. These reporter iPSC lines provide opportunity for monitoring and purifying these related cell lineages.

Presence and Characterization of a Novel cfr-Carrying Tn558 Transposon Derivative in Staphylococcus delphini Isolated From Retail Food

Antimicrobial resistance has become a major public health threat. Food-related Staphylococcus species have received much attention due to their multidrug resistance. The cfr gene associated with multidrug resistance has been consistently detected in food-derived Staphylococcus species. In this retrospective study, we examined the prevalence of cfr-positive Staphylococcus strains isolated from poultry meat in different geographical areas of China from 2011 to 2016. Two cfr-positive Staphylococcus delphini strains were identified from poultry meat in China. Comparative and whole-genome analyses were performed to characterize the genetic features and overall antimicrobial resistance genes in the two S. delphini isolates 245-1 and 2794-1. Whole-genome sequencing showed that they both harbored a novel 20,258-bp cfr-carrying Tn558 transposon derivative on their chromosomes. The Tn558 derivative harbors multiple antimicrobial resistance genes, including the transferable multiresistance gene cfr, chloramphenicol resistance gene fexA, aminoglycoside resistance genes aacA-aphD and aadD, and bleomycin resistance gene ble. Surprisingly, within the Tn558 derivative, an active unconventional circularizable structure containing various resistance genes and a copy of a direct repeat sequence was identified by two-step PCR. Furthermore, core genome phylogenetic analysis revealed that the cfr-positive S. delphini strains were most closely related to S. delphini 14S03313-1 isolated from Japan in 2017 and 14S03319-1 isolated from Switzerland in 2017. This study is the first report of S. delphini harboring a novel cfr-carrying Tn558 derivative isolated from retail food. This finding raises further concerns regarding the potential threat to food safety and public health safety. The occurrence and dissemination of similar cfr-carrying transposons from diverse Staphylococcus species need further surveillance.

Safety and efficacy of Nutrase P (6-phytase) for chickens for fattening, other poultry for fattening, reared for laying and ornamental birds.

Nutrase P is available in powder, granulated, thermostable granulate and liquid forms. It is based on a 6‐phytase produced by a genetically modified strain of K. phaffii. The production strain contains four copies of the ampicillin resistance gene and one copy of a bleomycin resistance gene. Although Nutrase PTS 10,000 (thermostable granulate) and Nutrase PG 10,000 (granulate) do not raise concern regarding the presence of viable cells of the production strain and its recombinant DNA, uncertainties remain on the presence of viable cells and DNA of the production strain in Nutrase PL 10,000 (liquid) and Nutrase PD 100,000 (powder). The FEEDAP Panel cannot conclude on the safety of the additive, in any form, for the target species for which application is made due to major limitations in the study in chickens for fattening provided. Considering the production strain and the results obtained in the genotoxicity studies, the Panel concludes that additive does not pose a toxicological safety concern regarding the consumers of the products obtained from animals receiving the additive. The additive, in its all formulations, is not a skin or eye irritant and is not dermal sensitiser. However, owing to the proteinaceous nature of the active substance it should be considered a respiratory sensitiser. The active substance of the additive is a protein and as such would not raise concerns for the environment. Considering that the production strain harbours AMR genes and there is uncertainties regarding the presence of viable cells and DNA of the production strain in Nutrase PL 10,000 (liquid) and Nutrase PD 100,000 (powder), the Panel cannot conclude on the safety of these two formulations of the additive for the target species, consumer, user and environment. Owing to the lack of data, the FEEDAP Panel cannot conclude on the efficacy of the additive.

Coexistence of bla NDM-1 and rmtC on a Transferrable Plasmid of a Novel ST192 Klebsiella aerogenes Clinical Isolate.

IntroductionThe occurrence and development of antibiotic resistance are mainly caused by the spread of large plasmids carrying multiple antibiotic resistance genes. Recently, the association between 16S rRNA methyltransferase genes and β-lactamase genes carried by the same plasmid is of concern.MethodsThe Klebsiella aerogenes 1564 was isolated from the catheter tip of a patient in a tertiary hospital, Shanghai, China. The presence of the blaNDM-1 and rmtC genes were assessed by PCR. Complete sequence of plasmid p1564 was determined. The K. aerogenes 1564 was characterized by antimicrobial susceptibility testing, Carbapenemase phenotype confirmation testing, conjugation experiment, S1-PFGE and multilocus sequence typing (MLST).ResultsHerein, we found that a New Delhi Metallo-β-lactamase-1 gene (blaNDM-1) and a 16S rRNA methyltransferase gene (rmtC) coexisted on a transferrable plasmid of a carbapenem-resistant K. aerogenes clinical isolate. The K. aerogenes clinical isolate was found to belong to a novel sequence type 192 (ST192) determined by MLST. The sequencing results of the plasmid p1564 carrying blaNDM-1 gene and rmtC gene showed that the size and guanine-cytosine content of the plasmid were 136, 902 bp and 51.8%, with 164 putative ORFs and two multidrug resistance gene islands. In addition to blaNDM-1and rmtC, the plasmid contained bleomycin resistance gene (bleMBL), CMY-6β-lactamase gene (blaCMY-6), quaternary ammonium compound resistance gene (sugE), truncated quaternary ammonium compound resistance gene (qacEΔ1), aminoglycoside resistance gene (aacA4) and sulfonamide resistance gene (sul1). By comparison, p1564 has high homology with pHS36-NDM from Salmonella enterica subsp. enterica serovar Stanley reported in China, with similar size and both belonging to plasmid incompatibility group A/C.ConclusionThe present study demonstrated for the first time the co-existence of rmtC and blaNDM-1 in a novel ST192 K. aerogenes. The spread of plasmids harboring both blaNDM-1 and rmtC may occur among Enterobacteriaceae in China.

Efficient expression of epidermal growth factor in Chlamydomonas reinhardtii CC400

The application of the green alga Chlamydomonas reinhardtii as a bioreactor is not adequate because of the difficulties caused by efficiency expressing foreign genes. To improve this efficiency a plasmid containing the epidermal growth factor (EGF) gene and a bleomycin resistance gene (ble) was constructed. We amplified the EGF gene according to the codon usage of C. reinhardtii. The vector carrying 2 expression cassettes for EGF gene and ble gene was constructed by adding rbc promoter and rbc terminator. Transformants, selected on Tris-acetate-phosphate medium containing 15 mg/L bleomycin, were screened by PCR and confirmed by Southern blotting, which showed that 3 transgenic C. reinhardtii cells contained only one copy of EGF gene integrated in different 3 sites of C. reinhardtii CC400 genome. Then EGF protein content of 3 transformants was determined by EGF precoated ELISA, indicating that EGF gene was first expressed, although at a low level, in algal cells. The presented study, as an example for expressing heterologous gene in green alga, provided feasibility to improve the efficiency of transformation of C. reinhardtii.

Detection of New Delhi Metallo-β-Lactamase Variants NDM-4, NDM-5, and NDM-7 in Enterobacter aerogenes Isolated from a Neonatal Intensive Care Unit of a North India Hospital: A First Report

A total 402 Enterobacteriaceae isolates were recovered from blood and rectal swabs of 1,000 infants admitted to the Neonatal Intensive Care Unit (NICU) of the Jawaharlal Medical College and Hospital Aligarh, India. Carbapenamase producers were determined by Carba NP phenotype biochemical assay. Out of 402 isolates, it was the first time three of the isolates were identified as Enterobacter aerogenes carrying blaNDM-4, blaNDM-5, and blaNDM-7 genes. These genes were identified by polymerase chain reaction (PCR) and sequence analysis. The isolates were further characterized to know the plasmid type and genetic environment features, including integron and IS elements. All the three E. aerogenes isolates (AK-93, AK-95, and AK-96) were resistant to all β-lactams, including carbapenems. The β-lactamase genes blaOXA-1, blaOXA-9, blaSHV-1, and blaVIM-2 were also found to be coassociated with blaNDM-4 in AK-93, blaOXA-1, blaOXA-9, and blaCMY-149 were found to be coexisted with blaNDM-5 in AK-95, and blaOXA-1; blaOXA-9, and blaCMY-145 were also found to be coassociated with blaNDM-7 in AK-96, identified by PCR analysis. Plasmid-based replicon typing revealed plasmids of different incompatibility in E. aerogenes in each of the isolates, AK-93 AK-95, and AK-96, respectively. ERIC-PCR was performed for the analysis of genetic relatedness of the strains. We found blaNDM-4, blaNDM-5, and blaNDM-7 producing three E. aerogenes strains, which were not clonally related. Genetic environment analysis revealed the presence of bleomycin resistance gene (bleMBL) to downstream of blaNDM and complete ISAba125 sequence were found upstream of blaNDM in all the three variants of these isolates. This is the first time we have identified blaNDM-4, blaNDM-5, and blaNDM-7 in E. aerogenes species, isolated from the NICU of a tertiary care hospital in India.

Characterization of BRPMBL, the Bleomycin Resistance Protein Associated with the Carbapenemase NDM.

The metallo-β-lactamase NDM-1 is among the most worrisome resistance determinants and is spreading worldwide among Gram-negative bacilli. A bleomycin resistance gene, bleMBL, downstream of the blaNDM-1 gene has been associated with resistance almost systematically. Here, we characterized the corresponding protein, BRPMBL, conferring resistance to bleomycin, an antitumoral glycopeptide molecule. We have determined whether the expression of the blaNDM-1-bleMBL operon is inducible in the presence of carbapenems and/or bleomycin-like molecules using quantitative reverse transcription-PCR (qRT-PCR), determination of imipenem and zeocin MICs, and carbapenemase-specific activity assays. We showed that the blaNDM-1-bleMBL operon is constitutively expressed. Using electrophoretic mobility shift and DNA protection assays performed with purified glutathione S-transferase (GST)-BRPMBL, we demonstrated that BRPMBL is able to bind and sequester bleomycin-like molecules, thus preventing bleomycin-dependent DNA degradation. In silico modeling confirmed that the mechanism of action required the dimerization of the BRPMBL protein in order to sequester bleomycin and prevent DNA damage. BRPMBL acts specifically on bleomycin-like molecules since cloning and expression of bleMBL in Staphyloccoccus aureus did not confer cross-resistance to any other antimicrobial glycopeptides such as vancomycin and teicoplanin.

Characterization of a blaNDM‑1‑harboring plasmid from a Salmonellaenterica clinical isolate in China.

The plasmid-mediated transmission of antibiotic resistance genes has been reported to be involved in the development of antibiotic resistance in bacteria, and poses a serious threat for the success of bacterial infection treatment and human health worldwide. The present study used a 454 GS-FLX pyrosequencing system to determine the ~140 kb nucleotide sequence of plasmid pHS36-NDM, which was identified in a Salmonella Stanley isolate from the stool sample of an 11-month-old girl at Lishui Central Hospital, China, and which contains a New Delhi metallo-β-lactamase-1 (NDM-1) carbapenem resistance gene (blaNDM-1). The 181 open reading frames encode proteins with functions including replication, stable inheritance, antibiotic resistance and mobile genetic elements. Both horizontal transfer and passage stability-related genes were identified in pHS36-NDM, including a conserved type 4 secretion system and stbA (stable plasmid inheritance protein A). Two multidrug resistance gene islands were identified: The ISEcp1-blaCMY transposition unit which contains a CMY-6 β-lactamase gene (blaCMY-6) and a quaternary ammonium compound resistance gene (sugE); and the intI1-ISCR27 accessory region, which contained a trimethoprim resistance gene (dfrA12), two aminoglycoside resistance genes (aadA2 and rmtC), a truncated quaternary ammonium compound resistance gene (qacE∆1), a sulfonamide resistance gene (sul1), the blaNDM-1 carbapenemase and a bleomycin resistance gene (bleMBL). pHS36-NDM shared high homology with other blaNDM-1-containing plasmids reported in Sweden, Italy and Japan. However, no previous international travel history was documented for the patient and her family, even to neighboring cities. Furthermore, pHS36-NDM is of a different incompatibility group to other published blaNDM-1-carrying plasmids reported in China, with low homology in the surrounding structure of blaNDM-1. The present study will facilitate the understanding of the underlying resistance and dispersal mechanism of pHS36-NDM, and will deepen our recognition of the ongoing spread of the blaNDM-1-containing plasmids.

Co-occurrence of NDM-5, OXA-181, and CMY-2 in a Clinical E. coli Isolate From a Patient in the United States

Background Carbapenem-resistant Enterobacteriacae (CRE) are an important public health problem. A mechanism of expansion of the CRE epidemic is transcontinental spread of multidrug-resistant organisms. Here, we present a patient with an intra-abdominal abscess due to Escherichia coli with an unusual multidrug resistance phenotype who was admitted to a tertiary hospital in Houston, TX after treatment in a country from the Middle East.Methods Identification and antimicrobial susceptibility testing were performed by standard methods. Whole-genome sequencing (WGS) was performed on an Illumina platform, with resistance genes identified by ResFinder 2.1 and plasmids recognized using PlasmidFinder 1.3. Results. It is confirmed via PCR, S1 pulsed field gel electrophoresis, and Southern blotting. Carbapenemase activity of bacterial lysates was assayed using 50 µM imipenem in the presence of both EDTA and zinc.ResultsThe E. coli isolate was resistant to all β-lactams, including ceftazidime/avibactam. The organism belonged to ST410 and harbored blaNDM-5, blaOXA-181, blaCMY-2, and blaTEM-1B. The blaOXA-181 gene was located on an IncX3 plasmid of ca. 50 Kb in association with the ISEcp1 mobile element and the qnrS21 gene encoding quinolone resistance. The blaNDM-5 gene was located on an FIA/FIB plasmid of ~100 Kb, in association with ΔISAba125, and upstream of a putative bleomycin resistance gene, a conserved arrangement among NDM expressing Gram-negative organisms. Cell lysate assays showed decreasing carbapenemase activity with increasing concentrations of EDTA and an increase in activity with the addition of zinc, suggesting the NDM-5 metallo-β-lactamase is largely responsible for the observed carbapenemase activity. Comparison with plasmid sequences available suggested convergence of resistance determinants captured from a wide geographic area.Conclusion Plasmid-mediated spread of β-lactamases among Enterobacteriaceae is a rapidly evolving threat, with the introduction of NDM-5 and OXA-181 in the United States being a particularly disturbing development. Introduction of multidrug-resistant organisms from areas of high prevalence of resistance may change the landscape of antimicrobial resistance in the United States.Disclosures All authors: No reported disclosures.

Production of valuable compounds by molds and yeasts.

We are pleased to dedicate this paper to Dr Julian E Davies. Julian is a giant among microbial biochemists. He began his professional career as an organic chemistry PhD student at Nottingham University, moved on to a postdoctoral fellowship at Columbia University, then became a lecturer at the University of Manchester, followed by a fellowship in microbial biochemistry at Harvard Medical School. In 1965, he studied genetics at the Pasteur Institute, and 2 years later joined the University of Wisconsin in the Department of Biochemistry. He later became part of Biogen as Research Director and then President. After Biogen, Julian became Chair of the Department of Microbiology at the University of British Columbia in Vancouver, Canada, where he has contributed in a major way to the reputation of this department for many years. He also served as an Adjunct Professor at the University of Geneva. Among Julian’s areas of study and accomplishment are fungal toxins including α-sarcin, chemical synthesis of triterpenes, mode of action of streptomycin and other aminoglycoside antibiotics, biochemical mechanisms of antibiotic resistance in clinical isolates of bacteria harboring resistance plasmids, their origins and evolution, secondary metabolism of microorganisms, structure and function of bacterial ribosomes, antibiotic resistance mutations in yeast ribosomes, cloning of resistance genes from an antibiotic-producing microbe, gene cloning for industrial purposes, engineering of herbicide resistance in useful crops, bleomycin-resistance gene in clinical isolates of Staphylococcus aureus and many other topics. He has been an excellent teacher, lecturing in both English and French around the world, and has organized international courses. Julian has also served on the NIH study sections, as Editor for several international journals, and was one of the founders of the journal Plasmid. We expect the impact of Julian’s accomplishments to continue into the future.

Transformation of Ulva mutabilis (Chlorophyta) by vector plasmids integrating into the genome

A method for the stable transformation of the green marine macroalga Ulva mutabilis was developed based on vector plasmids integrating into the genome. By combination of the expression signals (promoter, enhancer, and transcriptional termination sequences) of a chromosomal rbcS gene from U.mutabilis with the bleomycin resistance gene (ble) from Streptoalloteichus hindustanus, a dominant selectable marker gene was constructed for the preparation of a series of E.coli-U.mutabilis shuttle vector plasmids. Special vectors were prepared for the introduction and expression of foreign genes in Ulva, for insertional mutagenesis and gene tagging by plasmid integration into the genome, and for protein tagging by the green fluorescent protein, as well as tools for posttranscriptional gene silencing and cosmid cloning to prepare genomic gene libraries for mutant gene complementation. The vectors were successfully tested in pilot experiments, where they were efficiently introduced into Ulva gametes, zoospores or protoplasts of somatic blade cells by treatment with Ca(2+) -ions and polyethylene glycol under isotonic conditions at low ionic strength. The parthenogenetically propagated phleomycin-resistant transformants of the mutant slender (sl) and the wildtype (wt) were demonstrated to be carrying the plasmids randomly integrated into the chromosomes often as tandem repeat clusters.

New Delhi Metallo-β-Lactamase 1(NDM-1), the Dominant Carbapenemase Detected in Carbapenem-Resistant Enterobacter cloacae from Henan Province, China

The emergence of New Delhi metallo-β-lactamase 1 (NDM-1) has become established as a major public health threat and represents a new challenge in the treatment of infectious diseases. In this study, we report a high incidence and endemic spread of NDM-1-producing carbapenem-resistant Enterobacter cloacae isolates in Henan province, China. Eight (72.7%) out of eleven non-duplicated carbapenem-resistant E. cloacae isolates collected between June 2011 and May 2013 were identified as NDM-1 positive. The bla NDM-1 gene surrounded by an entire ISAba125 element and a bleomycin resistance gene ble MBL in these isolates were carried by diverse conjugatable plasmids (IncA/C, IncN, IncHI2 and untypeable) ranging from ~55 to ~360 kb. Molecular epidemiology analysis revealed that three NDM-1-producing E. cloacae belonged to the same multilocus sequence type (ST), ST120, two of which were classified as extensively drug-resistant (XDR) isolates susceptible only to tigecycline and colistin. The two XDR ST120 E. cloacae isolates co-harbored bla NDM-1, armA and fosA3 genes and could transfer resistance to carbapenems, fosfomycin and aminoglycosides simultaneously via a conjugation experiment. Our study demonstrated NDM-1 was the most prevalent metallo-β-lactamase (MBL) among carbapenem-resistant E.cloacae isolates and identified a potential endemic clone of ST120 in Henan province. These findings highlight the need for enhanced efforts to monitor the further spread of NDM-1 and XDR ST120 E. cloacae in this region.

New developments of RNAi in Paracoccidioides brasiliensis: prospects for high-throughput, genome-wide, functional genomics.

BackgroundThe Fungal Genome Initiative of the Broad Institute, in partnership with the Paracoccidioides research community, has recently sequenced the genome of representative isolates of this human-pathogen dimorphic fungus: Pb18 (S1), Pb03 (PS2) and Pb01. The accomplishment of future high-throughput, genome-wide, functional genomics will rely upon appropriate molecular tools and straightforward techniques to streamline the generation of stable loss-of-function phenotypes. In the past decades, RNAi has emerged as the most robust genetic technique to modulate or to suppress gene expression in diverse eukaryotes, including fungi. These molecular tools and techniques, adapted for RNAi, were up until now unavailable for P. brasiliensis.Methodology/Principal FindingsIn this paper, we report Agrobacterium tumefaciens mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150±24 yeast cell transformants per 1×106 viable yeast cells were obtained. Our approach is based on a bifunctional selective marker fusion protein consisted of the Streptoalloteichus hindustanus bleomycin-resistance gene (Shble) and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in P. brasiliensis. We also report successful GP43 gene knock-down through the expression of intron-containing hairpin RNA (ihpRNA) from a Gateway-adapted cassette (cALf) which was purpose-built for gene silencing in a high-throughput manner. Gp43 transcript levels were reduced by 73.1±22.9% with this approach.Conclusions/SignificanceWe have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future Paracoccidioides spp. functional genomics research.

Molecular Characterization of bla NDM-1 in a Sequence Type 235 Pseudomonas aeruginosa Isolate from France

An NDM-1 carbapenemase-producing Pseudomonas aeruginosa isolate was recovered from a patient hospitalized in France after a previous hospitalization in Serbia. Genetic studies revealed that the blaNDM-1 gene was surrounded by insertion sequence ISAba125 and a truncated bleomycin resistance gene. This blaNDM-1 region was a part of the variable region of a new complex class 1 integron bearing IS common region 1 (ISCR1). The presence of ISPa7 upstream of this integron suggests insertion in a chromosomally located Tn402-like structure.