Journal articles on the topic 'Cold hardy genes'

Gene expression for cold hardiness was investigated in a number of interspecific or intergeneric hybrids and amphiploids of wheat (Triticum aestivum L. em. Thell. or T. turgidum L.) and other members of the tribe Triticeae to assess the potential of alien species as donors of cold-hardiness genes for the improvement of wheat. Thinopyrum ponticum (Agropyron elongatum) hybrids with nonhardy T. aestivum had cold-hardiness levels similar to that of the more hardy Thinopyrum parent. Hybrids of Triticum cylindricum and both hardy and nonhardy T. aestivum were intermediate in cold hardiness with a tendency toward greater hardiness than the parental mean. Cold hardiness of hybrids between T. aestivum and Thinopyrum intermedium (Agropyron intermedium) was also close to the parental midpoint. Cold hardiness of T. aestivum – Secale cereale hybrids was greater than the less hardy parent. In contrast, cold-hardiness genes were not expressed beyond the level of the wheat parent in amphiploids combining wheat and the very hardy diploid species Agropyron cristatum and Secale cereale. The cold-hardiness level was also poor in an amphiploid produced from two relatively hardy tetraploid species (T. turgidum and T. cylindricum). These observations indicate that changes in ploidy level, relative to the parents, may influence the cold-hardiness potential of an interspecific combination by affecting gene dosage and possibly cell size. Poor expression of cold-hardiness genes from very hardy diploid genomes also indicated some degree of suppression, or homoeoallelic dominance of wheat cold-hardiness genes in amphiploids. Therefore, the performance of an interspecific hybrid or amphiploid of wheat may not give an accurate indication of the potential of alien species as gene donors for the improvement of wheat cold hardiness.Key words: gene expression, Triticum sp., triticale, Thinopyrum sp., Agropyron sp., Secale cereale.

Poncirus trifoliata (L.) Raf. is a cold-hardy, interfertile Citrus relative able to tolerate temperatures as low as –26°C when cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus varieties. A cold-induced cDNA library was constructed by subtractive hybridisation of non-acclimated and 2-d cold-acclimated P. trifoliata seedlings and many genes induced in response to cold were identified. Two of these cDNAs, PI-B05 and PI-C10, were selected from this library for further characterisation. Full-length cDNA sequences of these genes were obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). Sequence analysis revealed that PI-B05 contained an apetala2 / ethylene response factor (AP2 / ERF) domain and showed homology with ERF proteins from other plants, some of which are involved in environmental stress-induced gene expression. PI-C10 contained both AP2 / ERF and B3 DNA binding domains and showed homology with other plant proteins in the RAV subfamily of the AP2 / ERF transcription factors, some of which are induced in response to cold and other environmental stresses. Expression patterns of these genes in cold-tolerant P. trifoliata and cold-sensitive pummelo [Citrus grandis (L.) Osb.] in response to cold and drought at different time points were analysed by northern blots. Expression analysis showed that both genes were induced in response to cold, but not under drought conditions in cold-hardy P. trifoliata. However, little or no expression of these genes was detected by northern analysis in cold-sensitive pummelo under cold or drought conditions. The sequence analysis and expression data indicated that these genes may play a role in cold-responsive gene expression in cold-hardy P. trifoliata and could possibly be used for improving cold tolerance in cold-sensitive citrus cultivars.

The excellent cold hardiness of rye (Secale cereale L.) makes it a potential source of genetic variability for the improvement of this character in related species. However, when rye is combined with common wheat (Triticum aestivum L.) to produce octaploid triticale (X Triticosecale Wittmack, ABDR genomes), the superior rye cold hardiness is not expressed. To determine if the D genome of hexaploid wheat might be responsible for this lack of expression, hexaploid triticales (ABR genomes) were produced and evaluated for cold hardiness. All hexaploid triticales had cold hardiness levels similar to their tetraploid wheat parents. Small gains in cold hardiness of less than 2 °C were found when very non-hardy wheats were used as parents. This similarity in expression of cold hardiness in both octaploid and hexaploid triticales indicates that the D genome of wheat is not solely, if at all, responsible for the suppression of rye cold hardiness genes. There appears to be either a suppressor(s) of the rye cold hardiness genes on the AB genomes of wheat, or the expression of diploid rye genes is reduced to a uniform level by polyploidy in triticale. The suppression, or lack of expression, of rye cold hardiness genes in a wheat background make it imperative that cold-hardy wheats be selected as parents for the production of hardy triticales.Key words: Triticale, Secale, winter wheat, cold hardiness, gene expression

Identification of low temperature–regulated gene expression in Pachysandra terminalis: Pachysandra terminalis is a cold-hardy, evergreen plant species. In order to identify molecular mechanism of cold tolerance of this plant species, seedlings with four fully expanded leaves were subjected to 4, 0, and –1 °C low temperature treatments. Low temperature–induced genes were identified from treated plants using cDNA differential display. The cDNA fragments were cloned onto PCR-trap vectors. Low temperature regulation of these genes was confirmed by reverse-northern blot. Sequence analysis has identified that these genes can be classified into three groups, stress-related, photosystem-related. Most of the genes cannot find matching sequences in the database. To further study the regulation of these genes by temperature fluctuation, the plants were treated at 4, 0, and 40 °C. Northern blot analysis showed that several clones showed increased expression after cold and heat shock. Previous cold treatment at 4 °C can negate the effect of heat shock on expression of these genes. Complete sequence of these genes is cloned from the cDNA library and their temporal regulation by environmental stresses is analyzed using real-time PCR.

Citrus sp. are important commercial fruit crops throughout the world that are occasionally devastated by subfreezing temperatures. Poncirus trifoliata (maximum freeze tolerance of -26°C) is a close relative of commercial Citrus sp. (maximum freeze tolerance of -10°C) that has been used in breeding programs to develop more cold-hardy genotypes and as a rootstock to enhance freeze tolerance of the scion. Species with greater freeze tolerance vary in gene expression during cold acclimating temperatures. mRNA differential display (DDRT-PCR) and quantitative relative RT-PCR were used to study down regulation of gene expression in intact P. trifoliata exposed to a gradual cold acclimation regime to enhance our understanding of the mechanism that makes this specie so freeze tolerant. Six down-regulated genes were isolated and sequenced. These down-regulated genes showed high homology to the following known genes: chlorophyll a/b binding protein, photosystem II OEC 23, carbonic anhydrase, tumor related protein, pyrrolidone-carboxylate peptid ase and β-galactosidase. Photoprotection and the global control of gene expression related to photosynthesis appear to be important mechanisms for cold acclimation of P. trifoliata. Key words: Differential display, down-regulated genes, Poncirus trifoliata, cold acclimation and quantitative relative RT-PCR

Rose is the most economically important ornamental plant. However, cold stress seriously affects the survival and regrowth of garden roses in northern regions. Cold acclimation was studied using two genotypes (Rosa wichurana and R. hybrida ‘Yesterday’) selected from a rose breeding program. During the winter season (November to April), the cold hardiness of stems, soluble sugar content, and expression of dehydrins and the related key genes in the soluble sugar metabolism were analyzed. ‘Yesterday’ is more cold-hardy and acclimated faster, reaching its maximum cold hardiness in December. R. wichurana is relatively less cold-hardy, only reaching its maximum cold hardiness in January after prolonged exposure to freezing temperatures. Dehydrin transcripts accumulated significantly during November–January in both genotypes. Soluble sugars are highly involved in cold acclimation, with sucrose and oligosaccharides significantly correlated with cold hardiness. Sucrose occupied the highest proportion of total soluble sugars in both genotypes. During November–January, downregulation of RhSUS was found in both genotypes, while upregulation of RhSPS was observed in ‘Yesterday’ and upregulation of RhINV2 was found in R. wichurana. Oligosaccharides accumulated from November to February and decreased to a significantly low level in April. RhRS6 had a significant upregulation in December in R. wichurana. This study provides insight into the cold acclimation mechanism of roses by combining transcription patterns with metabolite quantification.

There is wide variation in Citrus and related genera in tolerance to cold and salt stress. While Poncirus trifoliata (L.) Raf. is an important rootstock for cold regions, it is salt sensitive. C. grandis (L.) Osb., on the other hand, is cold sensitive, but is relatively salt hardy. We are attempting to map genes (quantitative trait loci, QTLs) influencing salt and cold tolerance in Cirrus, using a BC1 population from [C. grandis × (C. grandis × P. trifoliata)]. As a first step, 2 year old containerized replicates of individual BC1 progeny plants have been salinized with 30 mM NaCl over a 9 month period under greenhouse conditions. Growth response under saline conditions, as evaluated by plant height and node number, varied significantly between individual progeny. Concentrations of 11 macro- and micro-elements, including Na and Cl, in leaf and root tissues were also determined. Ultimately, this data will be analyzed in conjunction with our current linkage map of this population, which consists of more than 200 marker genes, in order to map QTLs for salt tolerance.

Loss of freeze tolerance, or deacclimation, is an integral part of winter survival in woody perennials because untimely mid-winter or spring thaws followed by a hard freeze can cause severe injury to dehardened tissues. This study was undertaken to investigate deacclimation kinetics, particularly the timing and speed, of five blueberry (Vaccinium L.) cultivars (`Bluecrop', `Weymouth', `Ozarkblue', `Tifblue', and `Legacy'), with different germplasm compositions and mid-winter bud hardiness levels, in response to an environmentally controlled temperature regime. Based upon bud cold hardiness evaluations in 2000 and 2001, `Tifblue', a Vaccinium ashei Reade cultivar, was one of the least hardy and the fastest to deacclimate; `Bluecrop', a predominantly V. corymbosum L. cultivar, was the most hardy and the slowest to deacclimate; and `Ozarkblue', a predominantly V. corymbosum cultivar but including southern species V. darrowi Camp. and V. ashei, was intermediate in speed of deacclimation. `Weymouth' (predominantly V. corymbosum) and `Legacy' (73.4% V. corymbosum and 25% V. darrowi) were slow to intermediate deacclimators. Deacclimation rates did not correlate strictly with mid-winter bud hardiness. Data suggest that the southern germplasm component V. ashei may be responsible for the observed faster deacclimation whereas both southern species, V. darrowi and V. ashei, may contribute genes for cold sensitivity. Strong positive correlations between stage of bud opening and bud cold hardiness existed in both years (r = 0.90 and 0.82 in 2000 and 2001 study, respectively). Previously identified major blueberry dehydrins, 65-, 60-, and 14-kDa, progressively decreased in their abundance during incremental dehardening in `Bluecrop', `Weymouth', and `Tifblue'. However, down-regulation of the 14-kDa dehydrin most closely mirrored the loss in cold hardiness during deacclimation, and, therefore, may be involved in regulation of bud dehardening. Because differences in deacclimation rate were clearly evident among the genotypes studied, rate of deacclimation of the flower buds of blueberry should be an important consideration in breeding to improve winter survival.

We analyzed the transcriptomes in the shoots of five-year-old ‘Soomee’ peach trees (Prunus persica) during cold acclimation (CA), from early CA (end of October) to late CA (middle of January), and deacclimation (DA), from late CA to late DA (middle of March), to identify the genes involved in cold hardiness. Cold hardiness of the shoots increased from early to late CA, but decreased from late CA to late DA, as indicated by decreased and increased the median lethal temperature (LT50), respectively. Transcriptome analysis identified 17,208 assembled transcripts during all three stages. In total, 1891 and 3008 transcripts were differentially expressed with a |fold change| > 2 (p < 0.05) between early and late CA, and between late CA and late DA, respectively. Among them, 1522 and 2830, respectively, were functionally annotated with gene ontology (GO) terms having a greater proportion of differentially expressed genes (DEGs) associated with molecular function than biological process or cellular component categories. The biochemical pathways best represented both periods from early to late CA and from late CA to late DA were ‘metabolic pathway’ and ‘biosynthesis of secondary metabolites’. We validated these transcriptomic results by performing reverse transcription quantitative polymerase chain reaction on the selected DEGs showing significant fold changes. The relative expressions of the selected DEGs were closely related to the LT50 values of the peach tree shoots: ‘Soomee’ shoots exhibited higher relative expressions of the selected DEGs than shoots of the less cold-hardy ‘Odoroki’ peach trees. Irrespective of the cultivar, the relative expressions of the DEGs that were up- and then down-regulated during CA, from early to late CA, and DA, from late CA to late DA, were more closely correlated with cold hardiness than those of the DEGs that were down- and then up-regulated. Therefore, our results suggest that the significantly up- and then down-regulated DEGs are associated with cold hardiness in peach tree shoots. These DEGs, including early light-induced protein 1, chloroplastic, 14-kDa proline-rich protein DC2.15, glutamate dehydrogenase 2, and triacylglycerol lipase 2, could be candidate genes to determine cold hardiness.

Abstract Analysis of the pedigrees of selected eastern United States freestone peach [Prunus persica (L.) Batsch] cultivars reveals high degrees of inbreeding and coancestry. Commonly used parents include ‘Admiral Dewey’, ‘Elberta’, ‘Halehaven’, ‘J.H. Hale’, ‘Rio Oso Gem’, and ‘St. John’. These cultivars and their progeny were used as parents primarily for superior fruit quality. Selection for fruit quality has led to the intensive use of relatively few cultivars and a restriction in freestone peach germplasm. Future progress in the development of high quality, cold hardy, disease- and insect-resistant cultivars will depend upon expansion of the germplasm base, and identification and interfusion of genes conferring desired tree and fruit characters into existing eastern United States peach germplasm.

Deacclimation response is an important part of reproductive success in woody perennials because late winter or early spring thaws followed by hard freezes can cause severe injury to dehardened flower buds. There is a need to develop more spring-frost tolerant cultivars for the blueberry (Vaccinium L.) industry. The identification of later or slower deacclimating genotypes could be useful in breeding for more spring-frost tolerant cultivars. This study was undertaken to investigate cold hardiness and deacclimation kinetics under field conditions for 12 Vaccinium (section Cyanococcus A. Gray) genotypes (the cultivars Bluecrop, Duke, Legacy, Little Giant, Magnolia, Northcountry, Northsky, Ozarkblue, Pearl River, Tifblue, and Weymouth; and a population of V. constablaei Gray) with different germplasm compositions and expected mid-winter bud hardiness levels. Examination of bud cold hardiness (BCH) vs. weeks of deacclimation over a 7-week period in 2 consecutive years (2002 and 2003) revealed clear genotypic differences in cold hardiness and timing and rate of deacclimation. Among cultivars, `Legacy' was the least cold hardy at initial evaluation, even less so than `Tifblue'. Regarding deacclimation kinetics, the weekly intervals with the largest losses (i.e., high rates of deacclimation) also varied among genotypes. For `Duke', the largest losses in BCH were detected at weeks 2 and 3, making it the earliest deacclimator. For `Bluecrop', `Ozarkblue', `Weymouth', `Tifblue', and `Legacy', the greatest losses in BCH were observed at weeks 3 and 4. For `Little Giant', `Magnolia', `Northcountry', `Northsky', and `Pearl River', losses in BCH were greatest at weeks 4 and 5, while for V. constablaei, losses were greatest at weeks 6 and 7, making it the latest deacclimator. Deacclimation kinetics were not correlated with mid-winter hardiness or chilling requirements in any fixed pattern. On the other hand, a strong positive correlation was found between BCH and stage of bud opening (r = 0.84). A comparison of timing of deacclimation with germplasm composition indicated that V. constablaei was particularly late to deacclimate. `Little Giant', a 50:50 hybrid of V. constablaei and V. ashei Reade, was nearly as late to deacclimate as the 100% V. constablaei selections. Thus, V. constablaei may be useful in breeding programs to contribute genes for late deacclimation, which should translate into greater spring frost tolerance, in addition to genes for mid-winter hardiness.

Field winterhardiness is a critical trait in rose cultivars (Rosa ×hybrida) grown in northern climates. Although the molecular basis of cold hardiness has been well documented in model organisms such as Arabidopsis thaliana, little is known about the genetics and mechanisms underlying winterhardiness in roses. This research aims to explore the genetic control of winterhardiness for application in breeding programs using quantitative trail loci (QTL) analysis in two biparental rose populations derived from cold-hardy roses of the Canadian Explorer Series Collection. Field winterhardiness was assessed as a complex trait with winter damage and regrowth recorded in multiyear and multilocation trials in Ontario and Saskatchewan, Canada. In addition, this research explored the relationship between field measurements and electrolyte leakage recorded under artificial conditions. Electrolyte leakage had limited utility for application in rose breeding programs as a substitute for field evaluation, but did enable identification of QTL associated with potential cold hardiness candidate genes. A QTL for electrolyte leakage mapped to a genomic region that harbors a CBF1-like transcription factor. A total of 14 QTLs associated with field winter damage and regrowth were discovered, and they explained between 11% and 37% of the observed phenotypic variance. Two QTL associated with winter damage and regrowth overlapped with a known QTL for black spot (Diplocarpon rosae) disease resistance, Rdr1, in an environment under high disease pressure. Due to the complexity of field winterhardiness and its direct reliance on intertwined factors, such as overall plant health, moisture status, snow cover, and period of prolonged sub-zero temperatures, field trials are the ultimate measurement of field winterhardiness. Transgressive segregation was observed for all traits, and it was most likely due to complementary gene action. Field winter damage and regrowth were highly heritable in single environments, but they were subject to genotype × environment interaction resulting from pest pressure and severe climatic conditions.

SUMMARYThree groups of genes, Vrn, Ppd and Eps, control life-cycle duration in wheat (Triticum aestivum L.). The duration of a developmental phase between two stages is important for freezing resistance, heading time, anthesis and ripening date as well as yield component generation. The aim of the current study was to assess the effect of Vrn-D1 on wheat development. The vernalization genes Vrn-A1, -B1, -D1, -B3, photoperiod gene Ppd-1 and candidate genes Mot1 and FtsH4 for Eps in ‘G883’, ‘Pumai 9’ and their offspring, a group of sister lines (SLs) derived from an advanced generation, were genotyped using specific molecular markers. All detected loci were the same in the SLs and their parents except the Vrn-D1 locus. Three developmental traits, spike differentiation process, heading date and final leaf number on the main stem, were characterized in three sowing date treatments in the field. When temperatures increased, cultivars/lines carrying the dominant Vrn-D1 gene entered each spike differentiation process faster than those with the recessive vrn-D1 in the same sowing date treatment. Lines carrying Vrn-D1 had smaller final leaf number on the main stem than those with vrn-D1, and the heading dates of the former were earlier than those of the latter, especially in the fourth treatment, sown on 23 February 2012. These data suggest that Vrn-D1 confers a spring habit on wheat and the vrn-D1 confers a cold, hardy winter habit. The Vrn-D1 alleles play very important roles in semi-winter and tender spring wheat cultivars, especially in warm weather in Henan, China. Regulating developmental traits by tracing Vrn-D1 and getting an ideal combination of Vrn alleles to accommodate different wheat zones is a key role for future wheat molecular breeding.

Ethylene plays a pivotal role in plant stress resistance and 1-aminocyclopropane-1-carboxylic acid synthase (ACS) is the rate-limiting enzyme in ethylene biosynthesis. Upland cotton (Gossypium hirsutum L.) is the most important natural fiber crop, but the function of ACS in response to abiotic stress has rarely been reported in this plant. We identified 18 GaACS, 18 GrACS, and 35 GhACS genes in Gossypium arboreum, Gossypium raimondii and Gossypium hirsutum, respectively, that were classified as types I, II, III, or IV. Collinearity analysis showed that the GhACS genes were expanded from diploid cotton by the whole-genome-duplication. Multiple alignments showed that the C-terminals of the GhACS proteins were conserved, whereas the N-terminals of GhACS10 and GhACS12 were different from the N-terminals of AtACS10 and AtACS12, probably diverging during evolution. Most type II ACS genes were hardly expressed, whereas GhACS10/GhACS12 were expressed in many tissues and in response to abiotic stress; for example, they were highly and hardly expressed at the early stages of cold and heat exposure, respectively. The GhACS genes showed different expression profiles in response to cold, heat, drought, and salt stress by quantitative PCR analysis, which indicate the potential roles of them when encountering the various adverse conditions, and provide insights into GhACS functions in cotton’s adaptation to abiotic stress.

Stony hard (SH) peach (Prunus persica L. Batsch) fruit does not release ethylene and has very firm and crisp flesh at ripening, both on- and off-tree. Long-term cold storage can induce ethylene production and a serious risk of chilling injury in SH peach fruit; however, the regulatory mechanism underlying ethylene production in stony hard peach is relatively unclear. In this study, we analyzed the phytohormone levels, fruit firmness, transcriptome, and lipidome changes in SH peach ‘Zhongtao 9’ (CP9) during cold storage (4 °C). The expression level of the ethylene biosynthesis gene PpACS1 and the content of ethylene in SH peach fruit were found to be upregulated during cold storage. A peak in ABA release was observed before the release of ethylene and the genes involved in ABA biosynthesis and degradation, such as zeaxanthin epoxidase (ZEP) and 8’-hydroxylase (CYP707A) genes, were specifically induced in response to low temperatures. Fruit firmness decreased fairly slowly during the first 20 d of refrigeration, followed by a sharp decline. Furthermore, the expression level of genes encoding cell wall metabolic enzymes, such as polygalacturonase, pectin methylesterase, expansin, galactosidase, and β-galactosidase, were upregulated only upon refrigeration, as correlated with the decrease in fruit firmness. Lipids belonging to 23 sub-classes underwent differential rearrangement during cold storage, especially ceramide (Cer), monoglycosylceramide (CerG1), phosphatidic acid (PA), and diacyglyceride (DG), which may eventually lead to ethylene production. Exogenous PC treatment provoked a higher rate of ethylene production. We suspected that the abnormal metabolism of ABA and cell membrane lipids promotes the production of ethylene under low temperature conditions, causing the fruit to soften. In addition, ERF transcription factors also play an important role in regulating lipid, hormone, and cell wall metabolism during long-term cold storage. Overall, the results of this study give us a deeper understanding of the molecular mechanism of ethylene biosynthesis during the postharvest storage of SH peach fruit under low-temperature conditions.

Besides its health and spoilage hazards, Escherichia coli is a process hygiene indicator for cheeses made from milk that has undergone heat treatment. Hence, its ability to persist in cheesemaking plant environment and equipment is important. In total, 120 samples from two producing plants were analysed and 72 E. coli isolates were obtained. The target was to find out whether there is a difference in heat-resistance between persistent and non-persistent E. coli strains. The strains were selected using macrorestriction analysis and recurrent detection in cheesemaking plants hereby: one strain persisting in brine for blue-veined cheeses, two strains persisting in brine for hard cheeses and one non-persistent strain from raw material. Their D(50)-values were 196; 417; 370 and 182 min, respectively, D(59)-values ranged from 20 to 32 min and z-values were 7.5; 6.6; 8.1 and 9.0 °C, respectively. The non-persistent strain was the least resistant to heating to 50 °C but it was not the least resistant generally. All tested strains were highly heat-resistant and carried genes of the heat resistance locus LHR1 and/or LHR2. Our results emphasise the need to screen for the presence of LHR genes and the occurrence of heat-resistant E. coli in cheese production where they could survive sub-pasteurisation temperatures and contaminate the manufacturing environment and finished products.

Acid resistance (AR) in Escherichia coli is an important trait that protects this microorganism from the deleterious effect of low-pH environments. Reports on biofilm formation in E. coli K12 showed that the genes participating in AR were differentially expressed. Herein, we investigated the relationship between AR genes, in particular those coding for specific transcriptional regulators, and their biofilm-forming ability at the phenotypic level. The latter was measured in 96-well plates by staining the bacteria attached to the well, following 24-hour growth under static conditions, with crystal violet. The growth conditions were as follows: Luria Bertani (LB) medium at neutral and acidic pH, at 37C or 25C. We observed that the three major transcriptional regulators of the AR genes (gadX, gadE, gadW) only marginally affected biofilm formation in E. coli. However, a striking and novel finding was the different abilities of all the tested E. coli strains to form a biofilm depending on the temperature and pH of the medium: LB, pH 7.4, strongly supported biofilm formation at 25C, with biofilm being hardly detectable at 37C. On the contrary, LB, pH 5.5, best supported biofilm formation at 37C. Moreover, we observed that when E. coli carried a plasmid, the presence of the plasmid itself affected the ability to develop a biofilm, typically by increasing its formation. This phenomenon varies from plasmid to plasmid, depends on growth conditions, and, to the best of our knowledge, remains largely uninvestigated.

ABSTRACT The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in Escherichia coli that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential E. coli genes. Amber stop codons were introduced as “tagalong” mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes frr, gcpE, lpxC, map, murA, ppa, and rpsA. We were unable to isolate an amber mutation in ftsZ. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in gcpE, which was bacteriostatic. The strain carrying an amber mutation in murA was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development.

The σS (RpoS) subunit of RNA polymerase in Escherichia coli is a key master regulator which allows this bacterial model organism and important pathogen to adapt to and survive environmentally rough times. While hardly present in rapidly growing cells, σS strongly accumulates in response to many different stress conditions, partly replaces the vegetative sigma subunit in RNA polymerase and thereby reprograms this enzyme to transcribe σS-dependent genes (up to 10% of the E. coli genes). In this review, we summarize the extremely complex regulation of σS itself and multiple signal input at the level of this master regulator, we describe the way in which σS specifically recognizes “stress” promoters despite their similarity to vegetative promoters, and, while being far from comprehensive, we give a short overview of the far-reaching physiological impact of σS. With σS being a central and multiple signal integrator and master regulator of hundreds of genes organized in regulatory cascades and sub-networks or regulatory modules, this system also represents a key model system for analyzing complex cellular information processing and a starting point for understanding the complete regulatory network of an entire cell.

Food is an important vehicle for transmission of Shiga toxin–producing Escherichia coli (STEC). To assess the potential public health impact of STEC in Swiss raw milk cheese produced from cow's, goat's, and ewe's milk, 1,422 samples from semihard or hard cheese and 80 samples from soft cheese were examined for STEC, and isolated strains were further characterized. By PCR, STEC was detected after enrichment in 5.7% of the 1,502 raw milk cheese samples collected at the producer level. STEC-positive samples comprised 76 semihard, 8 soft, and 1 hard cheese. By colony hybridization, 29 STEC strains were isolated from 24 semihard and 5 soft cheeses. Thirteen of the 24 strains typeable with O antisera belonged to the serogroups O2, O22, and O91. More than half (58.6%) of the 29 strains belonged to O:H serotypes previously isolated from humans, and STEC O22:H8, O91:H10, O91:H21, and O174:H21 have also been identified as agents of hemolytic uremic syndrome. Typing of Shiga toxin genes showed that stx1 was only found in 2 strains, whereas 27 strains carried genes encoding for the Stx2 group, mainly stx2 and stx2vh-a/b. Production of Stx2 and Stx2vh-a/b subtypes might be an indicator for a severe outcome in patients. Nine strains harbored hlyA (enterohemorrhagic E. coli hemolysin), whereas none tested positive for eae (intimin). Consequently, semihard and hard raw milk cheese may be a potential source of STEC, and a notable proportion of the isolated non-O157 STEC strains belonged to serotypes or harbored Shiga toxin gene variants associated with human infections.

Bone regeneration after oral and maxillofacial surgery is a long-term process, which involves various mechanisms. Recently, cold atmospheric plasma (CAP) has become known to accelerate wound healing and have an antimicrobial effect. Since the use of CAP in dentistry is not yet established, the aim of the present study was to investigate the effect of CAP on human calvaria osteoblasts (HCO). HCO were treated with CAP for different durations of time and distances to the cells. Cell proliferation was determined by MTT assay and cell toxicity by LDH assay. Additionally, RT-qPCR was used to investigate effects on osteogenic markers, such as alkaline phosphatase (ALP), bone morphogenic protein (BMP)2, collagen (COL)1A1, osteonectin (SPARC), osteoprotegerin (OPG), osterix (OSX), receptor activator of NF-κB (RANK), RANK Ligand (RANKL), and Runt-related transcription factor (RUNX)2. There were small differences in cell proliferation and LDH release regarding treatment duration and distance to the cells. However, an increase in the expression of RANK and RANKL was observed at longer treatment times. Additionally, CAP caused a significant increase in mRNA expression of genes relevant to osteogenesis. In conclusion, CAP has a stimulating effect on osteoblasts and may thus represent a potential therapeutic approach in the regeneration of hard tissue defects.

Primary inoculum that survives overwintering is one of the key factors that determine the outbreak of plant disease. Pathogenic resting structures, such as chlamydospores, are an ideal inoculum for plant disease. Puzzlingly, Magnaporthe oryzae, a devastating fungal pathogen responsible for blast disease in rice, hardly form any morphologically changed resting structures, and we hypothesize that M. oryzae mainly relies on its physiological alteration to survive overwintering or other harsh environments. However, little progress on research into regulatory genes that facilitate the overwintering of rice blast pathogens has been made so far. Serine threonine protein kinase AGC/AKT, MoSch9, plays an important role in the spore-mediated pathogenesis of M. oryzae. Building on this finding, we discovered that in genetic and biological terms, MoSch9 plays a critical role in conidiophore stalk formation, hyphal-mediated pathogenesis, cold stress tolerance, and overwintering survival of M. oryzae. We discovered that the formation of conidiophore stalks and disease propagation using spores was severely compromised in the mutant strains, whereas hyphal-mediated pathogenesis and the root infection capability of M. oryzae were completely eradicated due to MoSch9 deleted mutants’ inability to form an appressorium-like structure. Most importantly, the functional and transcriptomic study of wild-type and MoSch9 mutant strains showed that MoSch9 plays a regulatory role in cold stress tolerance of M. oryzae through the transcription regulation of secondary metabolite synthesis, ATP hydrolyzing, and cell wall integrity proteins during osmotic stress and cold temperatures. From these results, we conclude that MoSch9 is essential for fungal infection-related morphogenesis and overwintering of M. oryzae.

We propose a simple model for interaction between gene candidates in the two strands of bacterial DNA (deoxyribonucleic acid). Our model assumes that ‘final’ genes appear in one of the two strands, that they do not overlap (in bacteria there is only a small percentage of overlap), and that the final genes maximize the occupancy rate, which is defined to be the proportion of the genome occupied by coding zones. We are more concerned with describing the organization and distribution of genes in bacterial DNA than with the very hard problem of identifying genes. To this end, an algorithm for selecting the final genes according to the previously outlined maximization criterion is proposed. We study the graphical and probabilistic properties of the model resulting from applying the maximization procedure to a Markovian representation of the genic and intergenic zones within the DNA strands, develop theoretical bounds on the occupancy rate (which, in our view, is a rather intractable quantity), and use the model to compute quantities of relevance to the Escherichia coli genome and compare these to annotation data. Although this work focuses on genomic modelling, we point out that the proposed model is not restricted to applications in this setting. It also serves to model other resource allocation problems.

ABSTRACT Cheese made with unpasteurized milk has been associated with outbreaks of illness. However, there are limited data on the prevalence of Shiga toxin–producing Escherichia coli (STEC) in these products and a lack of clarity over the significance of E. coli as a general indicator of hygiene in raw milk cheeses. The aim of this study was to provide further data to address both of these issues, as well as assessing the overall microbiological quality of raw milk cheeses available to consumers in England. A total of 629 samples of cheese were collected from retailers, catering premises, and manufacturers throughout England. The majority (80%) were made using cow's milk, with 14% made from sheep's milk and 5% from goat's milk. Samples were from 18 different countries of origin, with the majority originating from either the United Kingdom (40%) or France (35%). When interpreted against European Union microbiological criteria and United Kingdom guidance, 82% were considered to be of satisfactory microbiological quality, 5% were borderline, and 12% were unsatisfactory. Four samples (0.6%) were potentially injurious to health due to the isolation of STEC from one, &gt;104 CFU/g of coagulase-positive staphylococci in two, and &gt;100 CFU/g of Listeria monocytogenes in the fourth sample. Indicator E. coli and Listeria species were detected more frequently in soft compared with hard cheese. Higher levels of indicator E. coli were significantly associated with a greater likelihood of detecting Shiga toxin genes (stx1 and/or stx2). HIGHLIGHTS

Abstract. We identified genetic polymorphism in Mucin13 gene affecting E. coli adhesion patterns using (local isolate) diarrhoeagenic E. coli in Indian desi pigs. Five SNPs and one indel previously reported to be associated with enterotoxigenic E. coli (ETEC) F4ab/ac adhesion pattern were examined by designing PCR-RFLP protocol. The genotypic frequencies of only one SNP (g.22304A > G) differed significantly (at P≤0.05) in adhesive, non-adhesive and weakly adhesive population. The AA (306 sbp, 231 bp), AG (306, 231, 108, 198 bp) and GG (231, 198 bp, 108 b) genotypes of g.22304A > G locus were observed with frequencies 50.0 %, 21.25 % and 28.75 %, respectively and AG genotype was significantly (P≤0.05) associated with a non-adhesive pattern. The polymorphism information content of SNPs ranged from 17.67 (g.22124T > C) to 37.36 % (g.21471C > T) loci. Three loci (g.21471C > T, g.22124T > C and g.22304A > G) were significantly departed from Hardy–Weinberg equilibrium. The linkage disequilibrium analysis revealed locus g.22124T > C and g.22304A > G were significantly (P≤0.05) associated with each other. Expression profiling of target gene in jejuna of animals having AA, AG and GG genotypes revealed differences in various genotypes with the highest in the AA, moderate in the GG and low levels in the AG genotype, although they were statistically non-significant (at P≤0.05). The absence of significant effect of genotypes on MUC13 mRNA expression indicates no direct functional role, although the structural role can not be ignored as the putative receptor gene is located within targeted genomic region. Further, reports of same SNP association with an ETEC F4ab/ac adhesion pattern indicate the target gene's role in diarrhoea even caused by other strains of E. coli which is not ETEC.

Ulcerative colitis is a chronic inflammatory bowel disorder that is hard to cure once diagnosed. Bisdemethoxycurcumin has shown positive effects on inflammatory diseases. However, the underlying bioactive interaction between bisdemethoxycurcumin and ulcerative colitis is unclear. The objective of this study was to determine the core target and potential mechanism of action of bisdemethoxycurcumin as a therapy for ulcerative colitis. The public databases were used to identify potential targets for bisdemethoxycurcumin and ulcerative colitis. To investigate the potential mechanisms, the protein-protein interaction network, gene ontology analysis, and Kyoto encyclopedia of genes and genomes analysis have been carried out. Subsequently, experimental verification was conducted to confirm the findings. A total of 132 intersecting genes of bisdemethoxycurcumin, as well as ulcerative coli-tis-related targets, were obtained. SRC, EGFR, AKT1, and PIK3R1 were the targets of highest potential, and the PI3K/Akt and MAPK pathways may be essential for the treatment of ulcerative colitis by bisdemethoxycurcumin. Molecular docking demonstrated that bisdemethoxycurcumin combined well with SRC, EGFR, PIK3R1, and AKT1. Moreover, the in vitro experiments suggested that bisdemethoxycurcumin might reduce LPS-induced pro-inflammatory cytokines levels in RAW264.7 cells by suppressing PI3K/Akt and MAPK pathways. Our study provided a comprehensive overview of the potential targets and molecular mechanism of bisdemethoxycurcumin against ulcerative colitis. Furthermore, it also provided a theoretical basis for the clinical treatment of ulcerative colitis, as well as compelling evidence for further study on the mechanism of bisdemethoxycurcumin in the treatment of ulcerative colitis.

The oleaginous fungus Mortierella alpina is a safe source of polyunsaturated fatty acids (PUFA) in industrial food and feed production. Besides PUFA production, pharmaceutically relevant surface-active and antimicrobial oligopeptides were isolated from this basal fungus. Both production of fatty acids and oligopeptides rely on the biosynthesis and high turnover of branched-chain-amino acids (BCAA), especially l-leucine. However, the regulation of BCAA biosynthesis in basal fungi is largely unknown. Here, we report on the regulation of the leucine, isoleucine, and valine metabolism in M. alpina. In contrast to higher fungi, the biosynthetic genes for BCAA are hardly transcriptionally regulated, as shown by qRT-PCR analysis, which suggests a constant production of BCAAs. However, the enzymes of the leucine metabolism are tightly metabolically regulated. Three enzymes of the leucine metabolism were heterologously produced in Escherichia coli, one of which is inhibited by allosteric feedback loops: The key regulator is the α-isopropylmalate synthase LeuA1, which is strongly disabled by l-leucine, α-ketoisocaproate, and propionyl-CoA, the precursor of the odd-chain fatty acid catabolism. Its gene is not related to homologs from higher fungi, but it has been inherited from a phototrophic ancestor by horizontal gene transfer.

Pathogenic bacteria and viruses in medical environments can lead to treatment complications and hospital-acquired infections. Current disinfection protocols do not address hard-to-access areas or may be beyond line-of-sight treatment, such as with ultraviolet radiation. The COVID-19 pandemic further underscores the demand for reliable and effective disinfection methods to sterilize a wide array of surfaces and to keep up with the supply of personal protective equipment (PPE). We tested the efficacy of Sani Sport ozone devices to treat hospital equipment and surfaces for killing Escherichia coli, Enterococcus faecalis, Bacillus subtilis, and Deinococcus radiodurans by assessing Colony Forming Units (CFUs) after 30 min, 1 h, and 2 h of ozone treatment. Further gene expression analysis was conducted on live E. coli K12 immediately post treatment to understand the oxidative damage stress response transcriptome profile. Ozone treatment was also used to degrade synthetic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA as assessed by qPCR CT values. We observed significant and rapid killing of medically relevant and environmental bacteria across four surfaces (blankets, catheter, remotes, and syringes) within 30 min, and up to a 99% reduction in viable bacteria at the end of 2 h treatment cycles. RNA-seq analysis of E. coli K12 revealed 447 differentially expressed genes in response to ozone treatment and an enrichment for oxidative stress response and related pathways. RNA degradation of synthetic SARS-CoV-2 RNA was seen an hour into ozone treatment as compared to non-treated controls, and a non-replicative form of the virus was shown to have significant RNA degradation at 30 min. These results show the strong promise of ozone treatment of surfaces for reducing the risk of hospital-acquired infections and as a method for degradation of SARS-CoV-2 RNA.

Cancer stem cells (CSCs) are a small subset of cancer cells and are thought to play a critical role in the initiation and maintenance of tumor mass. CSCs exhibit similar hallmarks to normal stem cells, such as self-renewal, differentiation, and homeostasis. In addition, CSCs are equipped with several features so as to evade anticancer mechanisms. Therefore, it is hard to eliminate CSCs by conventional anticancer therapeutics that are effective at clearing bulk cancer cells. Interferons are innate cytokines and are the key players in immune surveillance to respond to invaded pathogens. Interferons are also crucial for adaptive immunity for the killing of specific aliens including cancer cells. However, CSCs usually evolve to escape from interferon-mediated immune surveillance and to shape the niche as a “cold” tumor microenvironment (TME). These CSC characteristics are related to their unique epigenetic regulations that are different from those of normal and bulk cancer cells. In this review, we introduce the roles of epigenetic modifiers, focusing on LSD1, BMI1, G9a, and SETDB1, in contributing to CSC characteristics and discussing the interplay between CSCs and interferon response. We also discuss the emerging strategy for eradicating CSCs by targeting these epigenetic modifiers, which can elevate cytosolic nuclei acids, trigger interferon response, and reshape a “hot” TME for improving cancer immunotherapy. The key epigenetic and immune genes involved in this crosstalk can be used as biomarkers for precision oncology.

AbstractAfter the first description of OXA-48 type carbapenemase, it has become endemic in Europe, Mediterranean and North African countries in a short time. OXA-48 carbapenemase is the most difficult type to determine and accurate diagnosis is crucial especially in endemic areas.The CarbaNP test was described as a rapid phenotypic evaluation method of carbapenemases activity. Sensitivity and specifity of this test were high within all carbapenemases genes. In our study, we evaluated the efficacy of CarbaNP test in routine laboratories located in an endemic area of OXA-48 producing Enterobacterales.A total of 53 Enterobacterales isolates were included in this study. Antimicrobial susceptibility of the isolates to imipenem, meropenem and ertapenem was determined. Polymerase Chain Reaction (PCR) was carried out for the detection of carbapenemases genes (blaKPC, blaNDM, blaBIC, blaIMP, blaVIM, blaSPM, blaAIM, blaDIM, blaGIM, blaSIM, and blaOXA-48). The Carba NP test was performed as in the protocol described previously.Altogether 31 isolates (58.4%) were blaOXA-48 positive (18 Klebsiella pneumoniae, 8 Escherichia coli, 2 Serratia marcescens, 1 Enterobacter aerogenes, 1 Pantoea agglomerans and 1 Morganella morganii). Among these isolates 3 (5.6%) and 2 (3.7%) isolates were also positive for blaVIM and blaSPM, respectively.The sensitivity and specifity of CarbaNP test were found 64.5, and 68.2% respectively. It was observed that determination of positive isolates is hard to distinguish and subjective.The CarbaNP test has suboptimal results and low of sensitivity and specifity for detection of OXA-48 producing Enterobacterales, and not suitable for detection of blaOXA-48 positive isolates in routine laboratories in endemic areas.

ABSTRACTThe food-borne pathogenListeria monocytogenesis known to colonize the lumen of the gallbladder in infected mice and to grow rapidly in this environment (J. Hardy et al., Science 303:851-853, 2004). However, relatively little is known about the mechanisms utilized by the pathogen to survive and grow in this location. We utilized gallbladder bile (GB bile) isolated directly from porcine gallbladders as anex vivomodel of gallbladder growth. We demonstrate that GB bile is generally nontoxic for bacteria and can readily support growth of a variety of bacterial species includingL. monocytogenes,Lactococcus lactis,Salmonella entericaserovar Typhimurium, andEscherichia coli. Significantly,L. monocytogenesgrew at the same rate as the nonpathogenic speciesListeria innocua, indicating that the pathogen does not possess specialized mechanisms that enable growth in this environment. However, when we reduced the pH of GB bile to pH 5.5 in order to mimic the release of bile within the small intestine, the toxicity of GB bile increased significantly and specific resistance mechanisms (Sigma B, BSH, and BilE) were essential for survival of the pathogen under these conditions. In order to identify genetic loci that are necessary for growth ofL. monocytogenesin the gallbladder, a mariner transposon bank was created and screened for mutants unable to replicate in GB bile. This led to the identification of mutants in six loci, including genes encoding enzymes involved in purine metabolism, amino acid biosynthesis, and biotin uptake. Although GB bile does not represent a significant impediment to bacterial growth, specific metabolic processes are required byL. monocytogenesin order to grow in this environment.

Both ionic and nanoparticle iron have been proposed as materials to control multidrug-resistant (MDR) bacteria. However, the potential bacteria to evolve resistance to nanoparticle bacteria remains unexplored. To this end, experimental evolution was utilized to produce five magnetite nanoparticle-resistant (FeNP1–5) populations of Escherichia coli. The control populations were not exposed to magnetite nanoparticles. The 24-h growth of these replicates was evaluated in the presence of increasing concentrations magnetite NPs as well as other ionic metals (gallium III, iron II, iron III, and silver I) and antibiotics (ampicillin, chloramphenicol, rifampicin, sulfanilamide, and tetracycline). Scanning electron microscopy was utilized to determine cell size and shape in response to magnetite nanoparticle selection. Whole genome sequencing was carried out to determine if any genomic changes resulted from magnetite nanoparticle resistance. After 25 days of selection, magnetite resistance was evident in the FeNP treatment. The FeNP populations also showed a highly significantly (p < 0.0001) greater 24-h growth as measured by optical density in metals (Fe (II), Fe (III), Ga (III), Ag, and Cu II) as well as antibiotics (ampicillin, chloramphenicol, rifampicin, sulfanilamide, and tetracycline). The FeNP-resistant populations also showed a significantly greater cell length compared to controls (p < 0.001). Genomic analysis of FeNP identified both polymorphisms and hard selective sweeps in the RNA polymerase genes rpoA, rpoB, and rpoC. Collectively, our results show that E. coli can rapidly evolve resistance to magnetite nanoparticles and that this result is correlated resistances to other metals and antibiotics. There were also changes in cell morphology resulting from adaptation to magnetite NPs. Thus, the various applications of magnetite nanoparticles could result in unanticipated changes in resistance to both metal and antibiotics.

Chicken litter application on land as an organic fertilizer is the cheapest and most environmentally safe method of disposing of the volume generated from the rapidly expanding poultry industry worldwide. However, little is known about the safety of chicken litter for land application and general release into the environment. Bridging this knowledge gap is crucial for maximizing the benefits of chicken litter as an organic fertilizer and mitigating negative impacts on human and environmental health. The key safety concerns of chicken litter are its contamination with pathogens, including bacteria, fungi, helminthes, parasitic protozoa, and viruses; antibiotics and antibiotic-resistant genes; growth hormones such as egg and meat boosters; heavy metals; and pesticides. Despite the paucity of literature about chicken litter safety for land application, the existing information was scattered and disjointed in various sources, thus making them not easily accessible and difficult to interpret. We consolidated scattered pieces of information about known contaminants found in chicken litter that are of potential risk to human, animal, and environmental health and how they are spread. This review tested the hypothesis that in its current form, chicken litter does not meet the minimum standards for application as organic fertilizer. The review entails a meta-analysis of technical reports, conference proceedings, peer-reviewed journal articles, and internet texts. Our findings indicate that direct land application of chicken litter could be harming animal, human, and environmental health. For example, counts of pathogenic strains of Eschericia coli (105–1010 CFU g−1) and Coliform bacteria (106–108 CFU g−1) exceeded the maximum permissible limits (MPLs) for land application. In Australia, 100% of broiler litter tested was contaminated with Actinobacillus and re-used broiler litter was more contaminated with Salmonella than non-re-used broiler litter. Similarly, in the US, all (100%) broiler litter was contaminated with Eschericia coli containing genes resistant to over seven antibiotics, particularly amoxicillin, ceftiofur, tetracycline, and sulfonamide. Chicken litter is also contaminated with a vast array of antibiotics and heavy metals. There are no standards set specifically for chicken litter for most of its known contaminants. Even where standards exist for related products such as compost, there is wide variation across countries and bodies mandated to set standards for safe disposal of organic wastes. More rigorous studies are needed to ascertain the level of contamination in chicken litter from both broilers and layers, especially in developing countries where there is hardly any data; set standards for all the contaminants; and standardize these standards across all agencies, for safe disposal of chicken litter on land.

Abstract This study evaluated the effects of phytogenics on performance, stool firmness, mortality and morbidity of weaned pigs housed in a commercial farm with historic incidence of post-weaning diarrhea associated with pathogenic E. coli. A total of 540 pigs (BW 7.34±0.9kg;) were used in a 42-day trial. Pigs were allotted by BW and sex into 30 pens (18pigs/pen). Pigs were fed a 2-phase feeding program, with each period being 14 and 28-d, respectively. Dietary treatments were: 1) Control and 2) Phytogenics (1,000 ppm Fresta®Protect, Delacon Biotechnik GmbH). Fecal score was determined daily by pen during the first 14-days (1=liquid diarrhea; 2=pasty feces; 3=formed feces; 4=well-formed feces; 5=hard feces). At day 42, fecal samples (n=15) were analyzed for copy numbers of the virulence factor genes fae (F-4 fimbria) and est-II (heat-stable toxin) by qPCR. Growth performance was not affected by treatments during phase 1. The ADG during phase 2 and overall period increased for pigs supplemented with phytogenics compared to control treatment (531 vs 501 g, P=0.015; 394 vs 375 g, P=0.025; respectively). Consequently, phytogenics tended to improve feed conversion during phase 2 and overall period compared to control treatment (1.48 vs 1.52, P=0.08; 1.46 vs 1.50, P=0.10). Phytogenics improved fecal score (3.8 vs 3.6, P&lt; 0.001), livability (97.4% vs 96%) and decreased the percentage of pigs that required medicine to control post-weaning diarrhea (1.8% vs 2.9%) compared to the control treatment. E. coli virulence factors in feces of animals supplemented with phytogenics were numerically lower than those fed the control treatment (8.5 vs 8.9 log10/g fae gene, P=0.32; 6.8 vs 7.2 log10/g est-II gene, P=0.50). Results suggest that the phytogenics used in this experiment is beneficial during post-weaning diarrhea outbreaks by improving performance and livability and by reducing the cost associated with medical treatments in weaned pigs

Abstract Background and Objectives Metallic antimicrobial materials are of growing interest due to their potential to control pathogenic and multidrug-resistant bacteria. Yet we do not know if utilizing these materials can lead to genetic adaptations that produce even more dangerous bacterial varieties. Methodology Here we utilize experimental evolution to produce strains of Escherichia coli K-12 MG1655 resistant to, the iron analog, gallium nitrate (Ga(NO3)3). Whole genome sequencing was utilized to determine genomic changes associated with gallium resistance. Computational modeling was utilized to propose potential molecular mechanisms of resistance. Results By day 10 of evolution, increased gallium resistance was evident in populations cultured in medium containing a sublethal concentration of gallium. Furthermore, these populations showed increased resistance to ionic silver and iron (III), but not iron (II) and no increase in traditional antibiotic resistance compared with controls and the ancestral strain. In contrast, the control populations showed increased resistance to rifampicin relative to the gallium-resistant and ancestral population. Genomic analysis identified hard selective sweeps of mutations in several genes in the gallium (III)-resistant lines including: fecA (iron citrate outer membrane transporter), insl1 (IS30 tranposase) one intergenic mutations arsC →/→ yhiS; (arsenate reductase/pseudogene) and in one pseudogene yedN ←; (iapH/yopM family). Two additional significant intergenic polymorphisms were found at frequencies &gt; 0.500 in fepD ←/→ entS (iron-enterobactin transporter subunit/enterobactin exporter, iron-regulated) and yfgF ←/→ yfgG (cyclic-di-GMP phosphodiesterase, anaerobic/uncharacterized protein). The control populations displayed mutations in the rpoB gene, a gene associated with rifampicin resistance. Conclusions This study corroborates recent results observed in experiments utilizing pathogenic Pseudomonas strains that also showed that Gram-negative bacteria can rapidly evolve resistance to an atom that mimics an essential micronutrient and shows the pleiotropic consequences associated with this adaptation. Lay summary We utilize experimental evolution to produce strains of Escherichia coli K-12 MG1655 resistant to, the iron analog, gallium nitrate (Ga(NO3)3). Whole genome sequencing was utilized to determine genomic changes associated with gallium resistance. Computational modeling was utilized to propose potential molecular mechanisms of resistance.

Abstract Benzenoids (C6–C1 aromatic compounds) play important roles in plant defense and are often produced upon herbivory. Black cottonwood (Populus trichocarpa) produces a variety of volatile and nonvolatile benzenoids involved in various defense responses. However, their biosynthesis in poplar is mainly unresolved. We showed feeding of the poplar leaf beetle (Chrysomela populi) on P. trichocarpa leaves led to increased emission of the benzenoid volatiles benzaldehyde, benzylalcohol, and benzyl benzoate. The accumulation of salicinoids, a group of nonvolatile phenolic defense glycosides composed in part of benzenoid units, was hardly affected by beetle herbivory. In planta labeling experiments revealed that volatile and nonvolatile poplar benzenoids are produced from cinnamic acid (C6–C3). The biosynthesis of C6–C1 aromatic compounds from cinnamic acid has been described in petunia (Petunia hybrida) flowers where the pathway includes a peroxisomal-localized chain shortening sequence, involving cinnamate-CoA ligase (CNL), cinnamoyl-CoA hydratase/dehydrogenase (CHD), and 3-ketoacyl-CoA thiolase (KAT). Sequence and phylogenetic analysis enabled the identification of small CNL, CHD, and KAT gene families in P. trichocarpa. Heterologous expression of the candidate genes in Escherichia coli and characterization of purified proteins in vitro revealed enzymatic activities similar to those described in petunia flowers. RNA interference-mediated knockdown of the CNL subfamily in gray poplar (Populus x canescens) resulted in decreased emission of C6–C1 aromatic volatiles upon herbivory, while constitutively accumulating salicinoids were not affected. This indicates the peroxisomal β-oxidative pathway participates in the formation of volatile benzenoids. The chain shortening steps for salicinoids, however, likely employ an alternative pathway.

The fatty acid β-oxidation multienzyme complex from Pseudomonas fragi, HDT, exhibits predominantly the three enzymic activities of 2-enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The HDT complex is encoded by the faoAB operon, consisting of the faoA and faoB genes that encode two individual constituents, the α-subunit and the β-subunit. We have constructed Escherichia coli overexpression systems for the faoAB gene product (coexpression of the α- and β-subunits), the α-subunit alone and the β-subunit alone, and have purified the three respective products. Gel-filtration analysis revealed that the faoAB gene product forms a heterotetrameric structure, α2β2, identical with the native HDT oligomeric state from P. fragi, whereas the α-subunit and β-subunit individually form dimers. Electron microscopy demonstrated that each protein morphologically adopts the above oligomeric structures. The HDT complex, reconstituted in vitro from the isolated α- and β-subunits, exhibits the three original enzymic activities and yields the same crystal as those from the native enzyme. CD measurements indicated that the α- and β-dimers hardly alter their global conformations upon the formation of the HDT complex. Interestingly, the β-dimer alone does not exhibit 3-oxoacyl-CoA thiolase activity, whereas the α-dimer alone exhibits both the 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities. These results suggest that the contact between the α- and β-subunits is essential for the thiolase activity. We have identified several structurally important proteolytic sites within each subunit, which are protected in the intact heterotetrameric molecule. These findings allow the possible location of the interface between the two subunits, which should be crucial for the exhibition of thiolase activity.

Abstract Background Gepotidacin (GEP) is a novel bacterial type II topoisomerase inhibitor in Phase 3 clinical trials for the treatment of gonorrhea and uncomplicated urinary tract infections (UTI). This study characterized fluoroquinolone (FQ)-not susceptible (not S) E. coli causing UTI in U.S. patients and evaluated the in vitro activity of GEP and comparators against various drug resistance (R) subsets. Methods 1,035 E. coli collected from 38 U.S. sites were included as part of the GEP Global UTI Surveillance Program (2019). Isolates were tested for susceptibility by broth microdilution. E. coli with MICs ≥0.5 mg/L for ciprofloxacin (not S) and/or ≥1 mg/L for levofloxacin (not S) were selected for screening of FQ-R mechanisms, and subjected to genome sequencing, followed by screening of FQ-R genes and QRDR mutations in GyrA, GyrB, ParC and ParE. Results A total of 26.8% (277/1,035) E. coli met the screening criteria for FQ-not S (Table). Overall, GEP had MIC90 values of 2 mg/L and 4 mg/L against FQ-S and FQ-not S isolates, respectively. Nitrofurantoin had activity against the FQ-S and FQ-not S subsets (98.8% and 94.2%S, respectively), whereas amoxicillin-clavulanate (86.5% and 59.6%S) and trimethoprim-sulfamethoxazole (75.8% and 37.0%S) had limited activity. Most FQ-not S isolates (52.7%; 146/277) had double mutations in GyrA and ParC, followed by those isolates (20.6%; 57/277) with double mutations in GyrA and single mutations in ParC and ParE. The third most common genotype was represented by isolates (14.8%;41/277) with double mutations in GyrA and a single mutation in ParC. GEP had MIC50 values of 1 mg/L or 2 mg/L and MIC90 values of 2 mg/L or 4 mg/L when tested against isolates with various combinations of QRDR mutations. 4.3% (12/277) of FQ-not S E. coli carried qnrB (6) or qnrS (6), and GEP (MIC50/90, 8/16 mg/L) had MICs of 0.5–32 mg/L against this subset. Conclusion GEP demonstrated potent activity against FQ-S and FQ-not S E. coli causing UTI in the U.S. In addition, GEP MIC did not seem to be affected by any combinations of FQ-R genes and QRDR mutations tested, except against the rare presence of qnrB/S genes. These data support the clinical development of GEP as a treatment option for UTI caused by FQ-S and FQ-not S E. coli isolates. Table Disclosures Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Timothy B. Doyle, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) S J Ryan Arends, PhD, AbbVie (formerly Allergan) (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Deborah Butler, n/a, GlaxoSmithKline, LLC (Employee) Nicole Scangarella-Oman, MS, GlaxoSmithKline, LLC (Employee) Jennifer M. Streit, BS, GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors (Individual(s) Involved: Self): Research Grant or Support; Allergan (Individual(s) Involved: Self): Research Grant or Support; Amicrobe, Inc (Individual(s) Involved: Self): Research Grant or Support; Amplyx Pharma (Individual(s) Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc. (Individual(s) Involved: Self): Research Grant or Support; Astellas (Individual(s) Involved: Self): Research Grant or Support; Basilea (Individual(s) Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center (Individual(s) Involved: Self): Research Grant or Support; BIDMC (Individual(s) Involved: Self): Research Grant or Support; bioMerieux Inc. (Individual(s) Involved: Self): Research Grant or Support; BioVersys Ag (Individual(s) Involved: Self): Research Grant or Support; Bugworks (Individual(s) Involved: Self): Research Grant or Support; Cidara (Individual(s) Involved: Self): Research Grant or Support; Cipla (Individual(s) Involved: Self): Research Grant or Support; Contrafect (Individual(s) Involved: Self): Research Grant or Support; Cormedix (Individual(s) Involved: Self): Research Grant or Support; Crestone, Inc. (Individual(s) Involved: Self): Research Grant or Support; Curza (Individual(s) Involved: Self): Research Grant or Support; CXC7 (Individual(s) Involved: Self): Research Grant or Support; Entasis (Individual(s) Involved: Self): Research Grant or Support; Fedora Pharmaceutical (Individual(s) Involved: Self): Research Grant or Support; Fimbrion Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Fox Chase (Individual(s) Involved: Self): Research Grant or Support; GlaxoSmithKline (Individual(s) Involved: Self): Research Grant or Support; Guardian Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Hardy Diagnostics (Individual(s) Involved: Self): Research Grant or Support; IHMA (Individual(s) Involved: Self): Research Grant or Support; Janssen Research & Development (Individual(s) Involved: Self): Research Grant or Support; Johnson & Johnson (Individual(s) Involved: Self): Research Grant or Support; Kaleido Biosceinces (Individual(s) Involved: Self): Research Grant or Support; KBP Biosciences (Individual(s) Involved: Self): Research Grant or Support; Luminex (Individual(s) Involved: Self): Research Grant or Support; Matrivax (Individual(s) Involved: Self): Research Grant or Support; Mayo Clinic (Individual(s) Involved: Self): Research Grant or Support; Medpace (Individual(s) Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd. (Individual(s) Involved: Self): Research Grant or Support; Melinta (Individual(s) Involved: Self): Research Grant or Support; Menarini (Individual(s) Involved: Self): Research Grant or Support; Merck (Individual(s) Involved: Self): Research Grant or Support; Meridian Bioscience Inc. (Individual(s) Involved: Self): Research Grant or Support; Micromyx (Individual(s) Involved: Self): Research Grant or Support; MicuRx (Individual(s) Involved: Self): Research Grant or Support; N8 Medical (Individual(s) Involved: Self): Research Grant or Support; Nabriva (Individual(s) Involved: Self): Research Grant or Support; National Institutes of Health (Individual(s) Involved: Self): Research Grant or Support; National University of Singapore (Individual(s) Involved: Self): Research Grant or Support; North Bristol NHS Trust (Individual(s) Involved: Self): Research Grant or Support; Novome Biotechnologies (Individual(s) Involved: Self): Research Grant or Support; Paratek (Individual(s) Involved: Self): Research Grant or Support; Pfizer (Individual(s) Involved: Self): Research Grant or Support; Prokaryotics Inc. (Individual(s) Involved: Self): Research Grant or Support; QPEX Biopharma (Individual(s) Involved: Self): Research Grant or Support; Rhode Island Hospital (Individual(s) Involved: Self): Research Grant or Support; RIHML (Individual(s) Involved: Self): Research Grant or Support; Roche (Individual(s) Involved: Self): Research Grant or Support; Roivant (Individual(s) Involved: Self): Research Grant or Support; Salvat (Individual(s) Involved: Self): Research Grant or Support; Scynexis (Individual(s) Involved: Self): Research Grant or Support; SeLux Diagnostics (Individual(s) Involved: Self): Research Grant or Support; Shionogi (Individual(s) Involved: Self): Research Grant or Support; Specific Diagnostics (Individual(s) Involved: Self): Research Grant or Support; Spero (Individual(s) Involved: Self): Research Grant or Support; SuperTrans Medical LT (Individual(s) Involved: Self): Research Grant or Support; T2 Biosystems (Individual(s) Involved: Self): Research Grant or Support; The University of Queensland (Individual(s) Involved: Self): Research Grant or Support; Thermo Fisher Scientific (Individual(s) Involved: Self): Research Grant or Support; Tufts Medical Center (Individual(s) Involved: Self): Research Grant or Support; Universite de Sherbrooke (Individual(s) Involved: Self): Research Grant or Support; University of Iowa (Individual(s) Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics (Individual(s) Involved: Self): Research Grant or Support; University of Wisconsin (Individual(s) Involved: Self): Research Grant or Support; UNT System College of Pharmacy (Individual(s) Involved: Self): Research Grant or Support; URMC (Individual(s) Involved: Self): Research Grant or Support; UT Southwestern (Individual(s) Involved: Self): Research Grant or Support; VenatoRx (Individual(s) Involved: Self): Research Grant or Support; Viosera Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Wayne State University (Individual(s) Involved: Self): Research Grant or Support

Abstract Abstract 2625 Background: It remains a huge challenge to observe fluorescent protein (GFP, RFP, etc.) gene-marked cells in bone, since bone is compact, poor lucency and porous, with different tissues such as vessels, nerve, cells, matrix and blood interlacing inside. However, it is very important to study the location, growth, migration and interaction of different stem cells and their offspring in bone. Aim: To better study different fluorescent protein gene-marked stem cells and microenvironment in bone, we establish a novel semi-solid decalcification (SSD) and research system. Methods: 1)Transplantation: Male BABL/C-GFP(H-2d) transgenic mice as donors and female FVB-RFP(H-2q) transgenic mice as recipients. Each RFP recipient mice were injected i.v. 5×10e6 allogeneic GFP bone marrow cells after 8 Gy TBI (n=10). Routinely, mice survival, weight, hemogram, GVHD manifestation were observed, with the fluorescence cells in peripheral blood and organs being traced. 2)Sections preparation: Total body perfusion fixation was performed with paraformaldehyde 21d after transplantation, and then different samples were collected for pathological examinations. The femurs were made frozen sections after semi-solid decalcification (SSD) system, while GMA plastic-embedding sections without decalcification, paraffin sections after EDTA decalcification, frozen sections after EDTA decalcification were also prepared as controls. Sections were observed by confocal microscopy. 3)Other researches: After SSD, observation and three-dimensional reconstruction were done by confocal microscopy; target tissue and cells were picked up for real-time quantified PCR for fluorescent protein genes and cell proliferation cytokines. Results: 1)Recipients RFP mice gained WBC recovery on (18.0±1.2)d, 90.0%±2.3% peripheral cells were GFP+ (n=10), 6 of 10 developed GVHD within 3m. 2)During SSD, hard component of the bone disappeared slowly, replaced gradually by semi-solid substance. SSD is even workable when the bone's diameter is large than 10cm. Frozen sections after SSD clearly showed unchanged position, form, and fluorescence of the GFP and RFP cells with repeatable hematoxylin and eosin(HE)and Wright-giemsa (RG) staining and immunohistochemical staining, fluorescence chromosomal in situ hybridization (FISH) after fluorescence observation and information from different tests of the same section can also be synthesized by computer. However, GMA cold embedding section could keep the cells where they are while losing the fluorescence, further more, embedding section only works well when the bone tissue is small (diameter<2mm). Frozen section after EDTA decalcification could keep the fluorescence with changed position and form during the progress. Paraffin sections can't keep neither the fluorescence nor the normal cell position and morphological characteristics. 3)Three-dimensional reconstruction shows the interesting relationships between different cells with different fluorescence and microenvironment by confocal microscopy. Quantified PCR described the cytokine expression profile of different fluorescence gene-marked cells. Conclusion: The SSD system shows great potency for the research of stem cells in vivo in bone while maintaining the morphological characteristics and structures between different cells without losing fluorescence signals. Another fantastic advantage is that a large number of techniques can be combined to our system to help us understand the homing, growth, proliferation, differentiation, migration and interaction of different target stem cells and their offspring. Disclosures: No relevant conflicts of interest to declare.

Abstract Background ST131 Escherichia coli possess virulence genes, adaptability for human colonization and are often associated to resistance genes. We evaluated the prevalence, O-antigen and susceptibility profiles of ST131 among β-lactam resistant E. coli isolates collected in US hospitals. Methods A total of 6,768 E. coli isolates collected during 2017 and 2018 were susceptibility tested using reference broth microdilution method. Among these, 1,154 displayed MIC values &gt;1 mg/L against 2 of the following: ceftazidime, ceftriaxone, cefepime or aztreonam or resistance to imipenem or meropenem (CLSI breakpoints) and were submitted to whole genome sequencing (WGS) and data analysis (MLST and O antigen). The genes wzx, wzy, wzm, and wzt were used to identify the O-antigen. Reference guided assembly for gene cluster O-AGC was used to differentiate O25a and O25b. Results Among the WGS E. coli isolates, 627 (54.3%) belonged to ST131 or were single loci variants (SLVs; 1 allele difference). A total of 586 (93.5% of ST131 and 50.8% of sequenced isolates) belonged to the O25b serotype. The remaining 41 isolates belonged to serotypes O16 (40 isolates) or O153var1 (1). ST131 isolates and O25b isolates were considerably more resistant to fluoroquinolones (92.0%-93.3% and 94.7%-95.5%, respectively) when compared to the overall WGS isolate collection (75.6%-77.0%). ST131-O25b isolates were more resistant to 12 /16 antimicrobial agents analyzed, including all β-lactam agents (0.9%-12.7% more resistant), fluoroquinolones (34.5-41.0%), and aminoglycosides (1.2-37.3%). ST131 (49.0%) and ST131-O25b (51.5%) isolates had higher multi-drug resistant (MDR) rates compared to all E. coli isolates (7.3%), WGS isolates (41.4%), and isolates that did not carry these traits (32.4% for non-ST131 and 12.2% for non-O25b). Conclusion ST131 and ST131-O25b E. coli isolates were common among β-lactam resistant E. coli from US hospitals. These isolates were significantly more resistant than their counterparts, despite the elevated resistance rates of the overall WGS collection. ST131-O25b E. coli isolates have the potential to present a challenge for antimicrobial treatment. Specific therapies that are effective against these isolates should be investigated. Disclosures Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors (Individual(s) Involved: Self): Research Grant or Support; Allergan (Individual(s) Involved: Self): Research Grant or Support; Amicrobe, Inc (Individual(s) Involved: Self): Research Grant or Support; Amplyx Pharma (Individual(s) Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc. (Individual(s) Involved: Self): Research Grant or Support; Astellas (Individual(s) Involved: Self): Research Grant or Support; Basilea (Individual(s) Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center (Individual(s) Involved: Self): Research Grant or Support; BIDMC (Individual(s) Involved: Self): Research Grant or Support; bioMerieux Inc. (Individual(s) Involved: Self): Research Grant or Support; BioVersys Ag (Individual(s) Involved: Self): Research Grant or Support; Bugworks (Individual(s) Involved: Self): Research Grant or Support; Cidara (Individual(s) Involved: Self): Research Grant or Support; Cipla (Individual(s) Involved: Self): Research Grant or Support; Contrafect (Individual(s) Involved: Self): Research Grant or Support; Cormedix (Individual(s) Involved: Self): Research Grant or Support; Crestone, Inc. (Individual(s) Involved: Self): Research Grant or Support; Curza (Individual(s) Involved: Self): Research Grant or Support; CXC7 (Individual(s) Involved: Self): Research Grant or Support; Entasis (Individual(s) Involved: Self): Research Grant or Support; Fedora Pharmaceutical (Individual(s) Involved: Self): Research Grant or Support; Fimbrion Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Fox Chase (Individual(s) Involved: Self): Research Grant or Support; GlaxoSmithKline (Individual(s) Involved: Self): Research Grant or Support; Guardian Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Hardy Diagnostics (Individual(s) Involved: Self): Research Grant or Support; IHMA (Individual(s) Involved: Self): Research Grant or Support; Janssen Research & Development (Individual(s) Involved: Self): Research Grant or Support; Johnson & Johnson (Individual(s) Involved: Self): Research Grant or Support; Kaleido Biosceinces (Individual(s) Involved: Self): Research Grant or Support; KBP Biosciences (Individual(s) Involved: Self): Research Grant or Support; Luminex (Individual(s) Involved: Self): Research Grant or Support; Matrivax (Individual(s) Involved: Self): Research Grant or Support; Mayo Clinic (Individual(s) Involved: Self): Research Grant or Support; Medpace (Individual(s) Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd. (Individual(s) Involved: Self): Research Grant or Support; Melinta (Individual(s) Involved: Self): Research Grant or Support; Menarini (Individual(s) Involved: Self): Research Grant or Support; Merck (Individual(s) Involved: Self): Research Grant or Support; Meridian Bioscience Inc. (Individual(s) Involved: Self): Research Grant or Support; Micromyx (Individual(s) Involved: Self): Research Grant or Support; MicuRx (Individual(s) Involved: Self): Research Grant or Support; N8 Medical (Individual(s) Involved: Self): Research Grant or Support; Nabriva (Individual(s) Involved: Self): Research Grant or Support; National Institutes of Health (Individual(s) Involved: Self): Research Grant or Support; National University of Singapore (Individual(s) Involved: Self): Research Grant or Support; North Bristol NHS Trust (Individual(s) Involved: Self): Research Grant or Support; Novome Biotechnologies (Individual(s) Involved: Self): Research Grant or Support; Paratek (Individual(s) Involved: Self): Research Grant or Support; Pfizer (Individual(s) Involved: Self): Research Grant or Support; Prokaryotics Inc. (Individual(s) Involved: Self): Research Grant or Support; QPEX Biopharma (Individual(s) Involved: Self): Research Grant or Support; Rhode Island Hospital (Individual(s) Involved: Self): Research Grant or Support; RIHML (Individual(s) Involved: Self): Research Grant or Support; Roche (Individual(s) Involved: Self): Research Grant or Support; Roivant (Individual(s) Involved: Self): Research Grant or Support; Salvat (Individual(s) Involved: Self): Research Grant or Support; Scynexis (Individual(s) Involved: Self): Research Grant or Support; SeLux Diagnostics (Individual(s) Involved: Self): Research Grant or Support; Shionogi (Individual(s) Involved: Self): Research Grant or Support; Specific Diagnostics (Individual(s) Involved: Self): Research Grant or Support; Spero (Individual(s) Involved: Self): Research Grant or Support; SuperTrans Medical LT (Individual(s) Involved: Self): Research Grant or Support; T2 Biosystems (Individual(s) Involved: Self): Research Grant or Support; The University of Queensland (Individual(s) Involved: Self): Research Grant or Support; Thermo Fisher Scientific (Individual(s) Involved: Self): Research Grant or Support; Tufts Medical Center (Individual(s) Involved: Self): Research Grant or Support; Universite de Sherbrooke (Individual(s) Involved: Self): Research Grant or Support; University of Iowa (Individual(s) Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics (Individual(s) Involved: Self): Research Grant or Support; University of Wisconsin (Individual(s) Involved: Self): Research Grant or Support; UNT System College of Pharmacy (Individual(s) Involved: Self): Research Grant or Support; URMC (Individual(s) Involved: Self): Research Grant or Support; UT Southwestern (Individual(s) Involved: Self): Research Grant or Support; VenatoRx (Individual(s) Involved: Self): Research Grant or Support; Viosera Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Wayne State University (Individual(s) Involved: Self): Research Grant or Support Timothy B. Doyle, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Andrew P. Davis, BS, Bravos Biosciences (Research Grant or Support) Valerie Kantro, BS, Bravos Biosciences (Research Grant or Support) Christopher M. Rubino, Pharm.D., 3-V Biosciences (Grant/Research Support)Achogen (Grant/Research Support)Amplyx Pharmaceuticals, Inc. (Grant/Research Support)Arixa Pharmaceuticals (Grant/Research Support)Arsanis Inc. (Grant/Research Support)B. Braun Medical Inc. (Grant/Research Support)Basilea Pharmaceutica (Grant/Research Support)BLC USA (Research Grant or Support)Boston Pharmaceuticals (Grant/Research Support)Bravos Biosciences, LLC (Grant/Research Support, Other Financial or Material Support, member/owner)Cidara Therapeutics Inc. (Grant/Research Support)Cipla, USA (Grant/Research Support)Corcept Therapeutics (Grant/Research Support)Cumberland Pharmaceuticals (Grant/Research Support)Debiopharm International SA (Grant/Research Support)Discuva Limited (Grant/Research Support)Emerald Lake Technologies (Grant/Research Support)Enhanced Pharmacodynamics (Grant/Research Support)Entasis Therapeutics (Grant/Research Support)E-Scape Bio (Grant/Research Support)Genentech (Grant/Research Support)Geom Therapeutics, Inc. (Grant/Research Support)GlaxoSmithKline (Grant/Research Support)Hoffmann-La Roche (Grant/Research Support)Horizon Orphan LLC (Grant/Research Support)ICPD Biosciences, LLC (Grant/Research Support, Other Financial or Material Support, member/owner)Indalo Therapeutics (Grant/Research Support)Insmed Inc. (Grant/Research Support)Institute for Clinical Pharmacodynamics (Employee)Iterum (Grant/Research Support)KBP Biosciences USA (Grant/Research Support)Kyoto Biopharma, Inc. (Grant/Research Support)Matinas (Grant/Research Support)Meiji Seika Pharma Co., Ltd. (Grant/Research Support)Melinta Therapeutics (Grant/Research Support)Menarini Ricerche S.p.A. (Grant/Research Support)Merck & Co., Inc (Grant/Research Support)Mutabilis (Grant/Research Support)Nabriva Therapeutics AG (Grant/Research Support)Naeja-RGM Pharmaceuticals (Grant/Research Support)Nosopharm SAS (Grant/Research Support)Novartis Pharmaceuticals Corp. (Grant/Research Support)NuCana Biomed (Grant/Research Support)Paratek Pharmaceuticals, Inc. (Grant/Research Support)Polyphor, Ltd. (Grant/Research Support)Prothena Corporation (Grant/Research Support)PTC Therapeutics (Grant/Research Support)Rempex Pharmaceuticals (Grant/Research Support)Roche TCRC (Grant/Research Support)Sagimet (Grant/Research Support)scPharmaceuticals Inc. (Grant/Research Support)Scynexis (Grant/Research Support)Spero Therapeutics (Grant/Research Support)TauRx Therapeutics (Grant/Research Support)Tetraphase Pharmaceuticals (Grant/Research Support)Theravance Biopharma Pharmaceutica (Grant/Research Support)USCAST (Grant/Research Support)VenatoRx (Grant/Research Support)Vical Incorporated (Grant/Research Support)Wockhardt Bio AG (Grant/Research Support)Zavante Therapeutics (Grant/Research Support)Zogenix International (Grant/Research Support) Sujata M. Bhavnani, Pharm.D., M.S., FIDSA, 3-V Biosciences (Grant/Research Support)Achogen (Grant/Research Support)Amplyx Pharmaceuticals, Inc. (Grant/Research Support)Arixa Pharmaceuticals (Grant/Research Support)Arsanis Inc. (Grant/Research Support)B. Braun Medical Inc. (Grant/Research Support)Basilea Pharmaceutica (Grant/Research Support)BLC USA (Grant/Research Support)Boston Pharmaceuticals (Grant/Research Support)Bravos Biosciences, LLC (Grant/Research Support, Other Financial or Material Support, member/owner)Cidara Therapeutics Inc. (Grant/Research Support)Cipla, USA (Grant/Research Support)Corcept Therapeutics (Grant/Research Support)Cumberland Pharmaceuticals (Grant/Research Support)Debiopharm International SA (Grant/Research Support)Discuva Limited (Grant/Research Support)Emerald Lake Technologies (Grant/Research Support)Enhanced Pharmacodynamics (Grant/Research Support)Entasis Therapeutics (Grant/Research Support)E-Scape Bio (Grant/Research Support)Genentech (Grant/Research Support)Geom Therapeutics, Inc. (Grant/Research Support)GlaxoSmithKline (Grant/Research Support)Hoffmann-La Roche (Grant/Research Support)Horizon Orphan LLC (Grant/Research Support)ICPD Biosciences, LLC (Grant/Research Support, Other Financial or Material Support, member/owner)Indalo Therapeutics (Grant/Research Support)Insmed Inc. (Grant/Research Support)Institute for Clinical Pharmacodynamics (Employee)Iterum (Grant/Research Support)KBP Biosciences USA (Grant/Research Support)Kyoto Biopharma, Inc. (Grant/Research Support)Matinas (Grant/Research Support)Meiji Seika Pharma Co., Ltd. (Grant/Research Support)Melinta Therapeutics (Grant/Research Support)Menarini Ricerche S.p.A. (Grant/Research Support)Merck & Co., Inc (Grant/Research Support)Mutabilis (Grant/Research Support)Nabriva Therapeutics AG (Grant/Research Support)Naeja-RGM Pharmaceuticals (Grant/Research Support)Nosopharm SAS (Grant/Research Support)Novartis Pharmaceuticals Corp. (Grant/Research Support)NuCana Biomed (Grant/Research Support)Paratek Pharmaceuticals, Inc. (Grant/Research Support)Polyphor, Ltd. (Grant/Research Support)Prothena Corporation (Grant/Research Support)PTC Therapeutics (Grant/Research Support)Rempex Pharmaceuticals (Grant/Research Support)Roche TCRC (Grant/Research Support)Sagimet (Grant/Research Support)scPharmaceuticals Inc. (Grant/Research Support)Scynexis (Grant/Research Support)Spero Therapeutics (Grant/Research Support)TauRx Therapeutics (Grant/Research Support)Tetraphase Pharmaceuticals (Grant/Research Support)Theravance Biopharma Pharmaceutica (Grant/Research Support)USCAST (Grant/Research Support)VenatoRx (Grant/Research Support)Vical Incorporated (Grant/Research Support)Wockhardt Bio AG (Grant/Research Support)Zavante Therapeutics (Grant/Research Support)Zogenix International (Grant/Research Support) Paul G. Ambrose, Pharm.D., FIDSA, 3-V Biosciences (Grant/Research Support)Achogen (Grant/Research Support)Amplyx Pharmaceuticals, Inc. (Grant/Research Support)Arixa Pharmaceuticals (Grant/Research Support)Arsanis Inc. (Grant/Research Support)B. Braun Medical Inc. (Grant/Research Support)Basilea Pharmaceutica (Grant/Research Support)BLC USA (Grant/Research Support)Boston Pharmaceuticals (Grant/Research Support)Bravos Biosciences, LLC (Grant/Research Support, Other Financial or Material Support, member/owner)Cidara Therapeutics Inc. (Grant/Research Support)Cipla, USA (Grant/Research Support)Corcept Therapeutics (Grant/Research Support)Cumberland Pharmaceuticals (Grant/Research Support)Debiopharm International SA (Grant/Research Support)Discuva Limited (Research Grant or Support)Emerald Lake Technologies (Grant/Research Support)Enhanced Pharmacodynamics (Grant/Research Support)Entasis Therapeutics (Grant/Research Support)E-Scape Bio (Grant/Research Support)Genentech (Grant/Research Support)Geom Therapeutics, Inc. (Grant/Research Support)GlaxoSmithKline (Grant/Research Support)Hoffmann-La Roche (Grant/Research Support)Horizon Orphan LLC (Grant/Research Support)ICPD Biosciences, LLC (Grant/Research Support, Other Financial or Material Support, member/owner)Indalo Therapeutics (Grant/Research Support)Insmed Inc. (Grant/Research Support)Institute for Clinical Pharmacodynamics (Employee)Iterum (Grant/Research Support)KBP Biosciences USA (Grant/Research Support)Kyoto Biopharma, Inc. (Grant/Research Support)Matinas (Grant/Research Support)Meiji Seika Pharma Co., Ltd. (Grant/Research Support)Melinta Therapeutics (Grant/Research Support)Menarini Ricerche S.p.A. (Grant/Research Support)Merck & Co., Inc (Grant/Research Support)Mutabilis (Grant/Research Support)Nabriva Therapeutics AG (Grant/Research Support)Naeja-RGM Pharmaceuticals (Grant/Research Support)Nosopharm SAS (Grant/Research Support)Novartis Pharmaceuticals Corp. (Grant/Research Support)NuCana Biomed (Grant/Research Support)Paratek Pharmaceuticals, Inc. (Grant/Research Support)Polyphor, Ltd. (Grant/Research Support)Prothena Corporation (Grant/Research Support)PTC Therapeutics (Grant/Research Support)Rempex Pharmaceuticals (Grant/Research Support)Roche TCRC (Grant/Research Support)Sagimet (Grant/Research Support)scPharmaceuticals Inc. (Grant/Research Support)Scynexis (Grant/Research Support)Spero Therapeutics (Grant/Research Support)TauRx Therapeutics (Grant/Research Support)Tetraphase Pharmaceuticals (Grant/Research Support)Theravance Biopharma Pharmaceutica (Grant/Research Support)USCAST (Grant/Research Support)VenatoRx (Grant/Research Support)Vical Incorporated (Grant/Research Support)Wockhardt Bio AG (Grant/Research Support)Zavante Therapeutics (Grant/Research Support)Zogenix International (Grant/Research Support)

Abstract Background Tebipenem (TBP) is an oral carbapenem in clinical development for treating complicated urinary tract infections (UTIs), including pyelonephritis. This study investigates the epidemiology of E. coli (EC) causing UTI in U.S. patients and the activity of TBP and comparators against various subsets. Methods A total of 2,395 EC recovered from urine samples during the 2018-2020 STEWARD Surveillance Program were included. Isolates were collected from medical centers in all 9 US Census Regions and centrally tested by reference broth microdilution method. MIC interpretation was based on CLSI criteria. Isolates that met MIC criteria were subjected to genome sequencing, followed by screening of extended-spectrum β-lactamase (ESBL) genes and epidemiology typing (MLST). Results A total of 16.1%, 15.4% and 14.6% of EC met the ESBL screening criteria in 2018, 2019 and 2020, respectively. 269/360 (74.7%) carried blaCTX-M and 2/360 (0.6%) had blaSHV-12. blaCMY (33/360; 9.2%) was the most common cephalosporinase, followed by blaDHA (7/360; 1.9%). A CRE phenotype was noted in 1 isolate from New York, which carried blaKPC-2. Acquired genes were not detected in 56 strains. 50 ST types were noted in isolates that met the ESBL criteria screening, with the majority of isolates being ST131 (56.2%). 21 (6.7%) and 19 (6.0%) isolates belonged to ST38 and ST1193, respectively, followed by STs represented by 8 or less isolates. Among ST131, 56.5% carried blaCTX-M from group 1 and 35.6% had genes associated with group 9. Overall, TBP showed consistent MIC50 values throughout the subsets. ERT had activity (≥97.0% susceptible) against the various subsets; however, lower susceptibility rates (85.7-90.6%) were noted against isolates carrying plasmid AmpC. Other agents (ceftriaxone and cefazolin) had activity only against non-ESBL producers. Conclusion bla CTX-M comprised the majority of acquired genes detected among ESBL strains, which belonged mostly to ST131, emphasizing the expansion of this clone. TBP showed consistent activity against all subsets, regardless of resistance genotype or lineage. These data support the clinical development of TBP as a convenient oral treatment option for UTI caused by EC. Disclosures Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Timothy B. Doyle, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Ian A. Critchley, Ph.D., Spero Therapeutics (Employee, Shareholder) Nicole Cotroneo, Spero Therapeutics (Employee, Shareholder) Jennifer M. Streit, BS, GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors (Individual(s) Involved: Self): Research Grant or Support; Allergan (Individual(s) Involved: Self): Research Grant or Support; Amicrobe, Inc (Individual(s) Involved: Self): Research Grant or Support; Amplyx Pharma (Individual(s) Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc. (Individual(s) Involved: Self): Research Grant or Support; Astellas (Individual(s) Involved: Self): Research Grant or Support; Basilea (Individual(s) Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center (Individual(s) Involved: Self): Research Grant or Support; BIDMC (Individual(s) Involved: Self): Research Grant or Support; bioMerieux Inc. (Individual(s) Involved: Self): Research Grant or Support; BioVersys Ag (Individual(s) Involved: Self): Research Grant or Support; Bugworks (Individual(s) Involved: Self): Research Grant or Support; Cidara (Individual(s) Involved: Self): Research Grant or Support; Cipla (Individual(s) Involved: Self): Research Grant or Support; Contrafect (Individual(s) Involved: Self): Research Grant or Support; Cormedix (Individual(s) Involved: Self): Research Grant or Support; Crestone, Inc. (Individual(s) Involved: Self): Research Grant or Support; Curza (Individual(s) Involved: Self): Research Grant or Support; CXC7 (Individual(s) Involved: Self): Research Grant or Support; Entasis (Individual(s) Involved: Self): Research Grant or Support; Fedora Pharmaceutical (Individual(s) Involved: Self): Research Grant or Support; Fimbrion Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Fox Chase (Individual(s) Involved: Self): Research Grant or Support; GlaxoSmithKline (Individual(s) Involved: Self): Research Grant or Support; Guardian Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Hardy Diagnostics (Individual(s) Involved: Self): Research Grant or Support; IHMA (Individual(s) Involved: Self): Research Grant or Support; Janssen Research & Development (Individual(s) Involved: Self): Research Grant or Support; Johnson & Johnson (Individual(s) Involved: Self): Research Grant or Support; Kaleido Biosceinces (Individual(s) Involved: Self): Research Grant or Support; KBP Biosciences (Individual(s) Involved: Self): Research Grant or Support; Luminex (Individual(s) Involved: Self): Research Grant or Support; Matrivax (Individual(s) Involved: Self): Research Grant or Support; Mayo Clinic (Individual(s) Involved: Self): Research Grant or Support; Medpace (Individual(s) Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd. (Individual(s) Involved: Self): Research Grant or Support; Melinta (Individual(s) Involved: Self): Research Grant or Support; Menarini (Individual(s) Involved: Self): Research Grant or Support; Merck (Individual(s) Involved: Self): Research Grant or Support; Meridian Bioscience Inc. (Individual(s) Involved: Self): Research Grant or Support; Micromyx (Individual(s) Involved: Self): Research Grant or Support; MicuRx (Individual(s) Involved: Self): Research Grant or Support; N8 Medical (Individual(s) Involved: Self): Research Grant or Support; Nabriva (Individual(s) Involved: Self): Research Grant or Support; National Institutes of Health (Individual(s) Involved: Self): Research Grant or Support; National University of Singapore (Individual(s) Involved: Self): Research Grant or Support; North Bristol NHS Trust (Individual(s) Involved: Self): Research Grant or Support; Novome Biotechnologies (Individual(s) Involved: Self): Research Grant or Support; Paratek (Individual(s) Involved: Self): Research Grant or Support; Pfizer (Individual(s) Involved: Self): Research Grant or Support; Prokaryotics Inc. (Individual(s) Involved: Self): Research Grant or Support; QPEX Biopharma (Individual(s) Involved: Self): Research Grant or Support; Rhode Island Hospital (Individual(s) Involved: Self): Research Grant or Support; RIHML (Individual(s) Involved: Self): Research Grant or Support; Roche (Individual(s) Involved: Self): Research Grant or Support; Roivant (Individual(s) Involved: Self): Research Grant or Support; Salvat (Individual(s) Involved: Self): Research Grant or Support; Scynexis (Individual(s) Involved: Self): Research Grant or Support; SeLux Diagnostics (Individual(s) Involved: Self): Research Grant or Support; Shionogi (Individual(s) Involved: Self): Research Grant or Support; Specific Diagnostics (Individual(s) Involved: Self): Research Grant or Support; Spero (Individual(s) Involved: Self): Research Grant or Support; SuperTrans Medical LT (Individual(s) Involved: Self): Research Grant or Support; T2 Biosystems (Individual(s) Involved: Self): Research Grant or Support; The University of Queensland (Individual(s) Involved: Self): Research Grant or Support; Thermo Fisher Scientific (Individual(s) Involved: Self): Research Grant or Support; Tufts Medical Center (Individual(s) Involved: Self): Research Grant or Support; Universite de Sherbrooke (Individual(s) Involved: Self): Research Grant or Support; University of Iowa (Individual(s) Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics (Individual(s) Involved: Self): Research Grant or Support; University of Wisconsin (Individual(s) Involved: Self): Research Grant or Support; UNT System College of Pharmacy (Individual(s) Involved: Self): Research Grant or Support; URMC (Individual(s) Involved: Self): Research Grant or Support; UT Southwestern (Individual(s) Involved: Self): Research Grant or Support; VenatoRx (Individual(s) Involved: Self): Research Grant or Support; Viosera Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Wayne State University (Individual(s) Involved: Self): Research Grant or Support

Abstract Abstract 1071 Zinc finger nucleases (ZFNs) are custom-designed DNA binding proteins that produce DNA double-strand breaks (DSBs) at predetermined genomic sites, stimulating homology-directed repair in the presence of donor template by many orders of magnitude over the spontaneous rate. The ability to target specific genes with ZFN technology opens therapeutic opportunities for gene correction and selective gene silencing. Sickle cell anemia (SCA) is an ideal disease target because correction of the single gene β-globin mutation in patient-derived, autologous hematopoietic stem progenitor cells (HSPCs) promises to be curative. One of the barriers to ZFN-based gene correction is the lack of a nonviral delivery system that achieves bulk transport of the nucleases to hard-to-transfect target cells, such as embryonic and HSPCs. To address this challenge, we set out to develop a delivery platform that is (i) gentle to the cell, (ii) provides tunable delivery rates, and (iii) achieves improved spatio-temporal control of the nucleases. To reconcile these goals, we have explored a method for direct delivery of ZFNs as proteins by receptor-mediated endocytosis. We selected the transferrin receptor pathway as our lead candidate on the rationale that all nucleated cells, including HSPCs, must import elemental iron to remain viable under ex vivo culture conditions. To test the feasibility of this strategy, this initial work used a ZFN pair targeted against a model GFP transgene. We optimized expression by pilot scale fermentation in an Escherichia coli host-vector system and purification to homogeneity by serial chromatography. We conjugated the purified ZFNs to the iron carrier protein, transferrin (tf), using SPDP, an amine and sulfhydryl reactive heterobifunctional crosslinker. The resulting disulfide linkage is designed to undergo scission (“self-immolation”) upon entry into the intracellular reducing environment. In vitro DNA cleavage assays and surface plasmon resonance binding assays demonstrated that ZFNs remained competent for target sequence cleavage following conjugation, with only mild to quantitative impairment of activity. To analyze delivery in biological systems, we measured time- and dose-dependence of tf-mediated ZFN uptake in human osteosarcoma (U2OS 2–6–3) cells. ZFNs in DAPI stained cell nuclei were detected by indirect immunofluorescence and signal intensity was measured in projections of deconvolved depth coded z-stacks. Nuclear uptake of tf-ZFN protein occurred in >95% of cells, was dose-dependent and linear with time in the lower dose ranges, and reached saturation as early as 60 min. Importantly, maximal nuclear uptake was indistinguishable from ZFN plasmid treated cells. These results indicate that endocytic delivery of ZFNs readily traverses the cellular membrane, overcomes the potential hurdle of endosomal trapping, and targets the nucleus with high efficiency. To demonstrate gene targeting activity, we used the U2OS 2–6–3 cell assay which bears a tandem transgene array at a single locus that is cleavable by our GFP ZFNs. Cells were transfected with lacI-ECFP to mark the target locus, incubated with tf-ZFNs, fixed, and stained for 53BP1, a signaling protein that marks DSBs. Recruitment of 53BP1 to the target locus was observed in 13% (18/135) of tf-ZFN treated cells, whereas no recruitment (0/152) was observed in untreated cells. These findings demonstrate that tf-conjugated ZFNs retain cleavage activity after nuclear uptake in a significant percentage of cells. To determine whether the tf-ZFNs are capable of stimulating gene correction, we transfected primary mouse adult fibroblasts carrying a mutant GFP transgene with donor template, incubated with tf-ZFNs, and evaluated cells at 72 h for gene correction as evidenced by GFP expression. Flow cytometry revealed a gene correction rate of 1–2%, identical to ZFN plasmid transfected cells, demonstrating that the technology of shuttling ZFN proteins to the cell interior via the tf-receptor pathway can deliver bioactive ZFNs to the nuclear compartment, target specific gene sequences, and induce homology-directed repair in the presence of donor DNA. We are currently testing these methods in hematopoietic stem cells, with the ultimate goal of correcting the sickle globin allele. Toward this end, we plan to adapt these approaches for high-throughput transfer of ZFN proteins directly to the hematopoietic stem progenitor cell. Disclosures: No relevant conflicts of interest to declare.

Abstract Background Carbapenems are broadly used for the treatment of ESBL-producing Enterobacterales isolates. The use of these agents led to an increase of carbapenem resistance among Enterobacterales. Monitoring isolates that carry β-lactamases is important to understand their prevalence and susceptibility to clinically available antimicrobial agents. We evaluated the prevalence of β-lactamases and the activity of antimicrobial agents against 1,209 isolates collected in 69 US hospitals. Methods A total of 9,686 Enterobacterales isolates collected during 2019 were susceptibility (S) tested by reference broth microdilution methods. Isolates submitted to whole genome sequencing were: (1) Escherichia coli (EC) and Klebsiella pneumoniae (KPN; n=817) displaying MIC values ≥2 mg/L for at least 2 of the following β-lactams: ceftazidime, ceftriaxone, aztreonam, or cefepime; (2) Enterobacter cloacae (ECL) and Citrobacter spp. (CIT; n=351) displaying MIC values ≥16 mg/L for ceftazidime and/or ≥2 mg/L for cefepime; and (3) Enterobacterales (n=118) displaying elevated carbapenem (meropenem and/or imipenem) MIC results at &gt;1 mg/L. Results A total of 723 isolates harbored ESBL genes but did not carry carbapenemases. The most common ESBL gene was blaCTX-M-15 (n=516), followed by blaCTX-M-14 (n=153). Most of these isolates were EC (278/147 for blaCTX-M-15/blaCTX-M-14), but 220 KPN harbored blaCTX-M-15. A total of 302 EC and KPN isolates carried blaOXA-1. Among ECL and CIT, blaCTX-M-15 and SHV genes encoding ESBLs were noted among 18 and 18 isolates. Carbapenemase genes were noted among 77 isolates, including 65 blaKPC, 3 blaSME, 6 blaOXA-48-like, and 3 blaNDM. Ceftazidime-avibactam (CAZ-AVI) was the only agent active against all ESBL-producers that did not carry carbapenemases (Table). CAZ-AVI was active against 90.9% of the isolates producing carbapenemases. Isolates resistant to this combination included 3 NDM-producers and 1 isolate harboring blaKPC-31. Conclusion Enterobacterales isolates carrying ESBLs, mainly blaCTX-M-15, were very prevalent in this collection of US isolates. CAZ-AVI was very active against isolates tested, including isolates producing carbapenemases that displayed resistance to many comparator agents. Disclosures Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors (Individual(s) Involved: Self): Research Grant or Support; Allergan (Individual(s) Involved: Self): Research Grant or Support; Amicrobe, Inc (Individual(s) Involved: Self): Research Grant or Support; Amplyx Pharma (Individual(s) Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc. (Individual(s) Involved: Self): Research Grant or Support; Astellas (Individual(s) Involved: Self): Research Grant or Support; Basilea (Individual(s) Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center (Individual(s) Involved: Self): Research Grant or Support; BIDMC (Individual(s) Involved: Self): Research Grant or Support; bioMerieux Inc. (Individual(s) Involved: Self): Research Grant or Support; BioVersys Ag (Individual(s) Involved: Self): Research Grant or Support; Bugworks (Individual(s) Involved: Self): Research Grant or Support; Cidara (Individual(s) Involved: Self): Research Grant or Support; Cipla (Individual(s) Involved: Self): Research Grant or Support; Contrafect (Individual(s) Involved: Self): Research Grant or Support; Cormedix (Individual(s) Involved: Self): Research Grant or Support; Crestone, Inc. (Individual(s) Involved: Self): Research Grant or Support; Curza (Individual(s) Involved: Self): Research Grant or Support; CXC7 (Individual(s) Involved: Self): Research Grant or Support; Entasis (Individual(s) Involved: Self): Research Grant or Support; Fedora Pharmaceutical (Individual(s) Involved: Self): Research Grant or Support; Fimbrion Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Fox Chase (Individual(s) Involved: Self): Research Grant or Support; GlaxoSmithKline (Individual(s) Involved: Self): Research Grant or Support; Guardian Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Hardy Diagnostics (Individual(s) Involved: Self): Research Grant or Support; IHMA (Individual(s) Involved: Self): Research Grant or Support; Janssen Research & Development (Individual(s) Involved: Self): Research Grant or Support; Johnson & Johnson (Individual(s) Involved: Self): Research Grant or Support; Kaleido Biosceinces (Individual(s) Involved: Self): Research Grant or Support; KBP Biosciences (Individual(s) Involved: Self): Research Grant or Support; Luminex (Individual(s) Involved: Self): Research Grant or Support; Matrivax (Individual(s) Involved: Self): Research Grant or Support; Mayo Clinic (Individual(s) Involved: Self): Research Grant or Support; Medpace (Individual(s) Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd. (Individual(s) Involved: Self): Research Grant or Support; Melinta (Individual(s) Involved: Self): Research Grant or Support; Menarini (Individual(s) Involved: Self): Research Grant or Support; Merck (Individual(s) Involved: Self): Research Grant or Support; Meridian Bioscience Inc. (Individual(s) Involved: Self): Research Grant or Support; Micromyx (Individual(s) Involved: Self): Research Grant or Support; MicuRx (Individual(s) Involved: Self): Research Grant or Support; N8 Medical (Individual(s) Involved: Self): Research Grant or Support; Nabriva (Individual(s) Involved: Self): Research Grant or Support; National Institutes of Health (Individual(s) Involved: Self): Research Grant or Support; National University of Singapore (Individual(s) Involved: Self): Research Grant or Support; North Bristol NHS Trust (Individual(s) Involved: Self): Research Grant or Support; Novome Biotechnologies (Individual(s) Involved: Self): Research Grant or Support; Paratek (Individual(s) Involved: Self): Research Grant or Support; Pfizer (Individual(s) Involved: Self): Research Grant or Support; Prokaryotics Inc. (Individual(s) Involved: Self): Research Grant or Support; QPEX Biopharma (Individual(s) Involved: Self): Research Grant or Support; Rhode Island Hospital (Individual(s) Involved: Self): Research Grant or Support; RIHML (Individual(s) Involved: Self): Research Grant or Support; Roche (Individual(s) Involved: Self): Research Grant or Support; Roivant (Individual(s) Involved: Self): Research Grant or Support; Salvat (Individual(s) Involved: Self): Research Grant or Support; Scynexis (Individual(s) Involved: Self): Research Grant or Support; SeLux Diagnostics (Individual(s) Involved: Self): Research Grant or Support; Shionogi (Individual(s) Involved: Self): Research Grant or Support; Specific Diagnostics (Individual(s) Involved: Self): Research Grant or Support; Spero (Individual(s) Involved: Self): Research Grant or Support; SuperTrans Medical LT (Individual(s) Involved: Self): Research Grant or Support; T2 Biosystems (Individual(s) Involved: Self): Research Grant or Support; The University of Queensland (Individual(s) Involved: Self): Research Grant or Support; Thermo Fisher Scientific (Individual(s) Involved: Self): Research Grant or Support; Tufts Medical Center (Individual(s) Involved: Self): Research Grant or Support; Universite de Sherbrooke (Individual(s) Involved: Self): Research Grant or Support; University of Iowa (Individual(s) Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics (Individual(s) Involved: Self): Research Grant or Support; University of Wisconsin (Individual(s) Involved: Self): Research Grant or Support; UNT System College of Pharmacy (Individual(s) Involved: Self): Research Grant or Support; URMC (Individual(s) Involved: Self): Research Grant or Support; UT Southwestern (Individual(s) Involved: Self): Research Grant or Support; VenatoRx (Individual(s) Involved: Self): Research Grant or Support; Viosera Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Wayne State University (Individual(s) Involved: Self): Research Grant or Support Lalitagauri M. Deshpande, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Pfizer, Inc. (Research Grant or Support) Timothy B. Doyle, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Helio S. Sader, MD, PhD, FIDSA, AbbVie (formerly Allergan) (Research Grant or Support)Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Department of Health and Human Services (Research Grant or Support, Contract no. HHSO100201600002C)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support)

Abstract Background Meropenem-vaborbactam (MVB) is an important addition to the armamentarium to treat infections caused by carbapenem-resistant Enterobacterales (CREs). Carbapenemase-negative (CN) CRE isolates have been reported, but the activity of newer agents against these isolates is still not well understood. We evaluated the activity of MVB against CN-CRE collected during 6 years of surveillance in US hospitals. Methods A total of 27,968 Enterobacterales isolates collected in US hospitals from 2014-2019 were susceptibility tested by reference broth microdilution methods. Results were interpreted using CLSI 2020 breakpoints. CRE isolates were submitted to PCR/sequencing (2014-2015) or whole genome sequencing (WGS; 2016-2019) for characterization of carbapenemase genes. Isolates from 2016-2019 were evaluated for other beta-lactam resistance mechanisms. Results Among 357 (1.3% of all isolates) CRE isolates identified during 6 years of surveillance, 48 (13.4% of the CRE) isolates did not produce known carbapenemases. The CN-CRE collection included 7 bacterial, species, or species complex. The top four most common species in the collection were K. pneumoniae (16 isolates) followed by E. cloacae (9), E. coli (8), and K. aerogenes (8). MVB was the most active agent tested against these isolates, inhibiting 47/48 (97.9%) of the isolates tested. The only isolate displaying a resistant MIC for MVB was a P. mirabilis (MIC, 16 mg/L) collected in 2015. Meropenem alone inhibited only 2.1% of the isolates. Other beta-lactams inhibited 4.2 to 14.6% of the isolates. Among non-beta-lactam comparator agents, tigecycline and amikacin inhibited 93.8 and 91.7% of the isolates, respectively, when applying CLSI or US FDA breakpoints. A total of 89.6% of the isolates had intermediate colistin MIC values. Among the 27 isolates collected from 2016-2019 that were submitted to WGS, 15 harbored CTX-M encoding genes. K. aerogenes and E. cloacae isolates (3 each) overexpressed AmpC. OmpC/K36 was disrupted in 20 isolates and OmpF/K35 was disrupted in 8 isolates. Conclusion MVB displayed good activity against CN-CRE isolates from US hospitals. This combination agent could be a good option to treat infections caused by these isolates. Disclosures Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors (Individual(s) Involved: Self): Research Grant or Support; Allergan (Individual(s) Involved: Self): Research Grant or Support; Amicrobe, Inc (Individual(s) Involved: Self): Research Grant or Support; Amplyx Pharma (Individual(s) Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc. (Individual(s) Involved: Self): Research Grant or Support; Astellas (Individual(s) Involved: Self): Research Grant or Support; Basilea (Individual(s) Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center (Individual(s) Involved: Self): Research Grant or Support; BIDMC (Individual(s) Involved: Self): Research Grant or Support; bioMerieux Inc. (Individual(s) Involved: Self): Research Grant or Support; BioVersys Ag (Individual(s) Involved: Self): Research Grant or Support; Bugworks (Individual(s) Involved: Self): Research Grant or Support; Cidara (Individual(s) Involved: Self): Research Grant or Support; Cipla (Individual(s) Involved: Self): Research Grant or Support; Contrafect (Individual(s) Involved: Self): Research Grant or Support; Cormedix (Individual(s) Involved: Self): Research Grant or Support; Crestone, Inc. (Individual(s) Involved: Self): Research Grant or Support; Curza (Individual(s) Involved: Self): Research Grant or Support; CXC7 (Individual(s) Involved: Self): Research Grant or Support; Entasis (Individual(s) Involved: Self): Research Grant or Support; Fedora Pharmaceutical (Individual(s) Involved: Self): Research Grant or Support; Fimbrion Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Fox Chase (Individual(s) Involved: Self): Research Grant or Support; GlaxoSmithKline (Individual(s) Involved: Self): Research Grant or Support; Guardian Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Hardy Diagnostics (Individual(s) Involved: Self): Research Grant or Support; IHMA (Individual(s) Involved: Self): Research Grant or Support; Janssen Research & Development (Individual(s) Involved: Self): Research Grant or Support; Johnson & Johnson (Individual(s) Involved: Self): Research Grant or Support; Kaleido Biosceinces (Individual(s) Involved: Self): Research Grant or Support; KBP Biosciences (Individual(s) Involved: Self): Research Grant or Support; Luminex (Individual(s) Involved: Self): Research Grant or Support; Matrivax (Individual(s) Involved: Self): Research Grant or Support; Mayo Clinic (Individual(s) Involved: Self): Research Grant or Support; Medpace (Individual(s) Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd. (Individual(s) Involved: Self): Research Grant or Support; Melinta (Individual(s) Involved: Self): Research Grant or Support; Menarini (Individual(s) Involved: Self): Research Grant or Support; Merck (Individual(s) Involved: Self): Research Grant or Support; Meridian Bioscience Inc. (Individual(s) Involved: Self): Research Grant or Support; Micromyx (Individual(s) Involved: Self): Research Grant or Support; MicuRx (Individual(s) Involved: Self): Research Grant or Support; N8 Medical (Individual(s) Involved: Self): Research Grant or Support; Nabriva (Individual(s) Involved: Self): Research Grant or Support; National Institutes of Health (Individual(s) Involved: Self): Research Grant or Support; National University of Singapore (Individual(s) Involved: Self): Research Grant or Support; North Bristol NHS Trust (Individual(s) Involved: Self): Research Grant or Support; Novome Biotechnologies (Individual(s) Involved: Self): Research Grant or Support; Paratek (Individual(s) Involved: Self): Research Grant or Support; Pfizer (Individual(s) Involved: Self): Research Grant or Support; Prokaryotics Inc. (Individual(s) Involved: Self): Research Grant or Support; QPEX Biopharma (Individual(s) Involved: Self): Research Grant or Support; Rhode Island Hospital (Individual(s) Involved: Self): Research Grant or Support; RIHML (Individual(s) Involved: Self): Research Grant or Support; Roche (Individual(s) Involved: Self): Research Grant or Support; Roivant (Individual(s) Involved: Self): Research Grant or Support; Salvat (Individual(s) Involved: Self): Research Grant or Support; Scynexis (Individual(s) Involved: Self): Research Grant or Support; SeLux Diagnostics (Individual(s) Involved: Self): Research Grant or Support; Shionogi (Individual(s) Involved: Self): Research Grant or Support; Specific Diagnostics (Individual(s) Involved: Self): Research Grant or Support; Spero (Individual(s) Involved: Self): Research Grant or Support; SuperTrans Medical LT (Individual(s) Involved: Self): Research Grant or Support; T2 Biosystems (Individual(s) Involved: Self): Research Grant or Support; The University of Queensland (Individual(s) Involved: Self): Research Grant or Support; Thermo Fisher Scientific (Individual(s) Involved: Self): Research Grant or Support; Tufts Medical Center (Individual(s) Involved: Self): Research Grant or Support; Universite de Sherbrooke (Individual(s) Involved: Self): Research Grant or Support; University of Iowa (Individual(s) Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics (Individual(s) Involved: Self): Research Grant or Support; University of Wisconsin (Individual(s) Involved: Self): Research Grant or Support; UNT System College of Pharmacy (Individual(s) Involved: Self): Research Grant or Support; URMC (Individual(s) Involved: Self): Research Grant or Support; UT Southwestern (Individual(s) Involved: Self): Research Grant or Support; VenatoRx (Individual(s) Involved: Self): Research Grant or Support; Viosera Therapeutics (Individual(s) Involved: Self): Research Grant or Support; Wayne State University (Individual(s) Involved: Self): Research Grant or Support Timothy B. Doyle, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Valerie Kantro, n/a, Melinta Therapeutics, Inc. (Research Grant or Support) Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Dee Shortridge, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support)