Isogenic Strains Research Articles

Comparative Analysis of Catabolic and Anabolic Dehydroshikimate Dehydratases for 3,4-DHBA Production in Escherichia coli.

The production of 3,4-dihydroxybenzoic acid (3,4-DHBA or protocatechuate) is a relevant task owing to 3,4-DHBA’s pharmaceutical properties and its use as a precursor for subsequent synthesis of high value-added chemicals. The microbial production of 3,4-DHBA using dehydroshikimate dehydratase (DSD) (EC: 4.2.1.118) has been demonstrated previously. DSDs from soil-dwelling organisms (where DSD is involved in quinate/shikimate degradation) and from Bacillus spp. (synthesizing the 3,4-DHBA-containing siderophore) were compared in terms of the kinetic properties and their ability to produce 3,4-DHBA. Catabolic DSDs from Corynebacterium glutamicum (QsuB) and Neurospora crassa (Qa-4) had higher Km (1 and 0.6 mM, respectively) and kcat (61 and 220 s−1, respectively) than biosynthetic AsbF from Bacillus thuringiensis (Km~0.04 mM, kcat~1 s−1). Product inhibition was found to be a crucial factor when choosing DSD for strain development. AsbF was more inhibited by 3,4-DHBA (IC50~0.08 mM), and Escherichia coli MG1655 ΔaroE PlacUV5-asbFattφ80 strain provided only 0.2 g/L 3,4-DHBA in test-tube fermentation. Isogenic strains MG1655 ΔaroE PlacUV5-qsuBattφ80 and MG1655 ΔaroE PlacUV5-qa-4attφ80 expressing QsuB and Qa-4 with IC50 ~0.35 mM and ~0.64 mM, respectively, accumulated 2.7 g/L 3,4-DHBA under the same conditions.

QTLViewer: an interactive webtool for genetic analysis in the Collaborative Cross and Diversity Outbred mouse populations.

The Collaborative Cross and the Diversity Outbred mouse populations are related multiparental populations, derived from the same 8 isogenic founder strains. They carry >50 M known genetic variants, which makes them ideal tools for mapping genetic loci that regulate phenotypes, including physiological and molecular traits. Mapping quantitative trait loci requires statistical and computational training, which can present a barrier to access for some researchers. The QTLViewer software allows users to graphically explore Collaborative Cross and Diversity Outbred quantitative trait locus mapping and related analyses performed through the R/qtl2 package. Additionally, the QTLViewer website serves as a repository for published Collaborative Cross and Diversity Outbred studies, increasing the accessibility of these genetic resources to the broader scientific community.

A novel hexa-segmented dsRNA mycovirus confers hypovirulence in the phytopathogenic fungus Diaporthe pseudophoenicicola.

A novel hexa-segmented double-stranded RNA (dsRNA) mycovirus was isolated and characterized from the filamentous phytopathogenic fungus Diaporthe pseudophoenicicola and was named Diaporthe pseudophoenicicola chrysovirus 1 (DpCV1). The full-length cDNAs of dsRNA1-6 were 3335, 3030, 3039, 2980, 963, and 780bp, respectively. Sequence analysis indicated the presence of nine open reading frames (ORFs) in the DpCV1 genome. ORF1 in dsRNA1 putatively encoded the RNA-dependent RNA polymerase (RdRp) and ORF3 in dsRNA2 encoded a capsid protein (CP). The seven remaining ORFs, ORF2 in dsRNA2, ORF4 in dsRNA3, ORF6, seven in dsRNA4, ORF8 in dsRNA5, and ORF9 in dsRNA6, encoded proteins with unknown functions. Phylogenetic analysis revealed that DpCV1 is closely related to members of the cluster I group within the family Chrysoviridae but formed a separate clade. Importantly, all the six segments of DpCV1 were cured successfully through single spore isolation to obtain the isogenic virus-free strains. DpCV1 can confer hypovirulence to the fungal host of Diaporthe pseudophoenicicola. Compared with the virus-free strain, WC02 harbouring the DpCV1 is more sensitive to fungicide prochloraz. Furthermore, the cell wall of DpCV1 infected strain was loose and enlarged. This is the first report of a hexa-segmented tentative chrysovirus in D. pseudophoenicicola.

Carbon dioxide fixation via production of succinic acid from glycerol in engineered Saccharomyces cerevisiae

BackgroundThe microbial production of succinic acid (SA) from renewable carbon sources via the reverse TCA (rTCA) pathway is a process potentially accompanied by net-fixation of carbon dioxide (CO2). Among reduced carbon sources, glycerol is particularly attractive since it allows a nearly twofold higher CO2-fixation yield compared to sugars. Recently, we described an engineered Saccharomyces cerevisiae strain which allowed SA production in synthetic glycerol medium with a maximum yield of 0.23 Cmol Cmol−1. The results of that previous study suggested that the glyoxylate cycle considerably contributed to SA accumulation in the respective strain. The current study aimed at improving the flux into the rTCA pathway accompanied by a higher CO2-fixation and SA yield.ResultsBy changing the design of the expression cassettes for the rTCA pathway, overexpressing PYC2, and adding CaCO3 to the batch fermentations, an SA yield on glycerol of 0.63 Cmol Cmol−1 was achieved (i.e. 47.1% of the theoretical maximum). The modifications in this 2nd-generation SA producer improved the maximum biomass-specific glycerol consumption rate by a factor of nearly four compared to the isogenic baseline strain solely equipped with the dihydroxyacetone (DHA) pathway for glycerol catabolism. The data also suggest that the glyoxylate cycle did not contribute to the SA production in the new strain. Cultivation conditions which directly or indirectly increased the concentration of bicarbonate, led to an accumulation of malate in addition to the predominant product SA (ca. 0.1 Cmol Cmol−1 at the time point when SA yield was highest). Off-gas analysis in controlled bioreactors with CO2-enriched gas-phase indicated that CO2 was fixed during the SA production phase.ConclusionsThe data strongly suggest that a major part of dicarboxylic acids in our 2nd-generation SA-producer was formed via the rTCA pathway enabling a net fixation of CO2. The greatly increased capacity of the rTCA pathway obviously allowed successful competition with other pathways for the common precursor pyruvate. The overexpression of PYC2 and the increased availability of bicarbonate, the co-substrate for the PYC reaction, further strengthened this capacity. The achievements are encouraging to invest in future efforts establishing a process for SA production from (crude) glycerol and CO2.

A qnrD-Plasmid Promotes Biofilm Formation and Class 1 Integron Gene Cassette Rearrangements in Escherichia coli.

Bacteria within biofilms may be exposed to sub-minimum inhibitory concentrations (sub-MICs) of antibiotics. Cell-to-cell contact within biofilms facilitates horizontal gene transfers and favors induction of the SOS response. Altogether, it participates in the emergence of antibiotic resistance. Aminoglycosides at sub-MICs can induce the SOS response through NO accumulation in E. coli carrying the small plasmid with the quinolone resistance qnrD gene (pDIJ09-518a). In this study, we show that in E. coli pDIJ09-518a, the SOS response triggered by sub-MICs of aminoglycosides has important consequences, promoting genetic rearrangement in class 1 integrons and biofilm formation. We found that the integrase expression was increased in E. coli carrying pDIJ09-518a in the presence of tobramycin, which was not observed for the WT isogenic strain that did not carry the qnrD-plasmid. Moreover, we showed that biofilm production was significantly increased in E. coli WT/pDIJ09-518a compared to the WT strain. However, such a higher production was decreased when the Hmp-NO detoxification pathway was fully functional by overexpressing Hmp. Our results showing that a qnrD-plasmid can promote biofilm formation in E. coli and potentiate the acquisition and spread of resistance determinants for other antibiotics complicate the attempts to counteract antibiotic resistance and prevention of biofilm development even further. We anticipate that our findings emphasize the complex challenges that will impact the decisions about antibiotic stewardship, and other decisions related to retaining antibiotics as effective drugs and the development of new drugs.

Property space mapping of Pseudomonas aeruginosa permeability to small molecules

Two membrane cell envelopes act as selective permeability barriers in Gram-negative bacteria, protecting cells against antibiotics and other small molecules. Significant efforts are being directed toward understanding how small molecules permeate these barriers. In this study, we developed an approach to analyze the permeation of compounds into Gram-negative bacteria and applied it to Pseudomonas aeruginosa, an important human pathogen notorious for resistance to multiple antibiotics. The approach uses mass spectrometric measurements of accumulation of a library of structurally diverse compounds in four isogenic strains of P. aeruginosa with varied permeability barriers. We further developed a machine learning algorithm that generates a deterministic classification model with minimal synonymity between the descriptors. This model predicted good permeators into P. aeruginosa with an accuracy of 89% and precision above 58%. The good permeators are broadly distributed in the property space and can be mapped to six distinct regions representing diverse chemical scaffolds. We posit that this approach can be used for more detailed mapping of the property space and for rational design of compounds with high Gram-negative permeability.

Noninvasive assessment of gut function using transcriptional recording sentinel cells.

Transcriptional recording by CRISPR spacer acquisition from RNA endows engineered Escherichia coli with synthetic memory, which through Record-seq reveals transcriptome-scale records. Microbial sentinels that traverse the gastrointestinal tract capture a wide range of genes and pathways that describe interactions with the host, including quantitative shifts in the molecular environment that result from alterations in the host diet, induced inflammation, and microbiome complexity. We demonstrate multiplexed recording using barcoded CRISPR arrays, enabling the reconstruction of transcriptional histories of isogenic bacterial strains in vivo. Record-seq therefore provides a scalable, noninvasive platform for interrogating intestinal and microbial physiology throughout the length of the intestine without manipulations to host physiology and can determine how single microbial genetic differences alter the way in which the microbe adapts to the host intestinal environment.

The Alternative Sigma Factor SigL Influences Clostridioides difficile Toxin Production, Sporulation, and Cell Surface Properties.

The alternative sigma factor SigL (Sigma-54) facilitates bacterial adaptation to the extracellular environment by modulating the expression of defined gene subsets. A homolog of the gene encoding SigL is conserved in the diarrheagenic pathogen Clostridioides difficile. To explore the contribution of SigL to C. difficile biology, we generated sigL-disruption mutants (sigL::erm) in strains belonging to two phylogenetically distinct lineages—the human-relevant Ribotype 027 (strain BI-1) and the veterinary-relevant Ribotype 078 (strain CDC1). Comparative proteomics analyses of mutants and isogenic parental strains revealed lineage-specific SigL regulons. Concomitantly, loss of SigL resulted in pleiotropic and distinct phenotypic alterations in the two strains. Sporulation kinetics, biofilm formation, and cell surface-associated phenotypes were altered in CDC1 sigL::erm relative to the isogenic parent strain but remained unchanged in BI-1 sigL::erm. In contrast, secreted toxin levels were significantly elevated only in the BI-1 sigL::erm mutant relative to its isogenic parent. We also engineered SigL overexpressing strains and observed enhanced biofilm formation in the CDC1 background, and reduced spore titers as well as dampened sporulation kinetics in both strains. Thus, we contend that SigL is a key, pleiotropic regulator that dynamically influences C. difficile's virulence factor landscape, and thereby, its interactions with host tissues and co-resident microbes.

Engineering proton-coupled hexose uptake in Saccharomyces cerevisiae for improved ethanol yield.

BackgroundIn the yeast Saccharomyces cerevisiae, which is widely applied for industrial bioethanol production, uptake of hexoses is mediated by transporters with a facilitated diffusion mechanism. In anaerobic cultures, a higher ethanol yield can be achieved when transport of hexoses is proton-coupled, because of the lower net ATP yield of sugar dissimilation. In this study, the facilitated diffusion transport system for hexose sugars of S. cerevisiae was replaced by hexose–proton symport.ResultsIntroduction of heterologous glucose– or fructose–proton symporters in an hxt0 yeast background strain (derived from CEN.PK2-1C) restored growth on the corresponding sugar under aerobic conditions. After applying an evolutionary engineering strategy to enable anaerobic growth, the hexose–proton symporter-expressing strains were grown in anaerobic, hexose-limited chemostats on synthetic defined medium, which showed that the biomass yield of the resulting strains was decreased by 44.0-47.6%, whereas the ethanol yield had increased by up to 17.2% (from 1.51 to 1.77 mol mol hexose−1) compared to an isogenic strain expressing the hexose uniporter HXT5. To apply this strategy to increase the ethanol yield on sucrose, we constructed a platform strain in which all genes encoding hexose transporters, disaccharide transporters and disaccharide hydrolases were deleted, after which a combination of a glucose–proton symporter, fructose–proton symporter and extracellular invertase (SUC2) were introduced. After evolution, the resulting strain exhibited a 16.6% increased anaerobic ethanol yield (from 1.51 to 1.76 mol mol hexose equivalent−1) and 46.6% decreased biomass yield on sucrose.ConclusionsThis study provides a proof-of-concept for the replacement of the endogenous hexose transporters of S. cerevisiae by hexose-proton symport, and the concomitant decrease in ATP yield, to greatly improve the anaerobic yield of ethanol on sugar. Moreover, the sugar-negative platform strain constructed in this study acts as a valuable starting point for future studies on sugar transport or development of cell factories requiring specific sugar transport mechanisms.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-022-02145-7.

How Yeast Antifungal Resistance Gene Analysis Is Essential to Validate Antifungal Susceptibility Testing Systems.

ObjectivesThe antifungal susceptibility testing (AFST) of yeast pathogen alerts clinicians about the potential emergence of resistance. In this study, we compared two commercial microdilution AFST methods: Sensititre YeastOne read visually (YO) and MICRONAUT-AM read visually (MN) or spectrophotometrically (MNV), interpreted with Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing criteria, respectively.MethodsOverall, 97 strains from 19 yeast species were measured for nine antifungal drugs including a total of 873 observations. First, the minimal inhibitory concentration (MIC) was compared between YO and MNV, and between MNV and MN, either directly or by assigning them to five susceptibility categories. Those categories were based on the number of MIC dilutions around the breakpoint or epidemiological cut-off reference values (ECOFFs or ECVs). Second, YO and MNV methods were evaluated for their ability to detect the elevation of MICs due to mutation in antifungal resistance genes, thanks to pairs or triplets of isogenic strains isolated from a single patient along a treatment previously analyzed for antifungal resistance gene mutations. Reproducibility measurement was evaluated, thanks to three quality control (QC) strains.ResultsYO and MNV direct MIC comparisons obtained a global agreement of 67%. Performing susceptibility category comparisons, only 22% and 49% of the MICs could be assigned to categories using breakpoints and ECOFFs/ECVs, respectively, and 40% could not be assigned due to the lack of criteria in both consortia. The YO and MN susceptibility categories gave accuracies as low as 50%, revealing the difficulty to implement this method of comparison. In contrast, using the antifungal resistance gene sequences as a gold standard, we demonstrated that both methods (YO and MN) were equally able to detect the acquisition of resistance in the Candida strains, even if MN showed a global lower MIC elevation than YO. Finally, no major differences in reproducibility were observed between the three AFST methods.ConclusionThis study demonstrates the valuable use of both commercial microdilution AFST methods to detect antifungal resistance due to point mutations in antifungal resistance genes. We highlighted the difficulty to conduct conclusive analyses without antifungal gene sequence data as a gold standard. Indeed, MIC comparisons taking into account the consortia criteria of interpretation remain difficult even after the effort of harmonization.

Tumorigenic mechanisms of estrogen and Helicobacter pylori cytotoxin-associated gene A in estrogen receptor α-positive diffuse-type gastric adenocarcinoma.

Diffuse-type gastric cancer (DGC), for which Helicobacter pylori infection is a causal factor, is associated with poor prognosis among young women, possibly due to female hormones such as estrogen. We aimed to identify the carcinogenesis induced by estrogen and H. pylori in DGC. We screened and selected estrogen receptor alpha (ERα)-positive (MKN45) and ERα-negative (SNU5) DGC cell lines. H. pylori strain 60190 and its isogenic mutant strain lacking cytotoxin-associated gene A (60190ΔCagA) were used to infect MKN45 cells. And the cytotoxin-related gene A (CagA) cDNA which was cloned into pSP65-SR-HA (cagA-pSP65SRa) vector was used to transfect MKN45 cells. Tumor samples were used for DGC organoid culture. In MKN45 cells, we found that estradiol promotes epithelial-mesenchymal transition (EMT) and stemness phenotypes via HOTAIR expression. These effects were further enhanced by the addition of CagA secreted by H. pylori but were reversed by co-treatment with fulvestrant (ICI 182,780), a selective ER degrader. We also validated the effect of estrogen on DGC organoids. ERα expression was associated with tumor invasion and HOTAIR expression in DGC patients with overt H. pylori infection. These findings may explain the rapid DGC progression in young women with physiologically high levels of estrogen and suggest that fulvestrant with ovarian function suppression could serve as a tumor-suppressive agent in premenopausal patients with DGC.

Interplay between Amoxicillin Resistance and Osmotic Stress in Helicobacter pylori.

Rising antibiotic resistance rates are a growing concern for all pathogens, including Helicobacter pylori. We previously examined the association of specific mutations in PBP1 with amoxicillin resistance and fitness in H. pylori and found that V374L and N562Y mutations were associated with resistance, but also resulted in fitness defects. Furthermore, we found that hyperosmotic stress differentially altered the fitness of strains bearing these mutations; survival of the V374L strain was decreased by hyperosmotic stress, but the N562Y strain showed increased cell survival relative to that of wild-type G27. The finding that amoxicillin-resistant strains show environmentally dictated changes in fitness suggests a previously unexplored interaction between amoxicillin resistance and osmotic stress in H. pylori. Here, we further characterized the interaction between osmotic stress and amoxicillin resistance. Wild-type and isogenic PBP1 mutant strains were exposed to amoxicillin, various osmotic stressors, or combined antibiotic and osmotic stress, and viability was monitored. While subinhibitory concentrations of NaCl did not affect H. pylori viability, the combination of NaCl and amoxicillin resulted in synergistic killing; this was true even for the antibiotic-resistant strains. Moreover, similar synergy was found with other beta-lactams, but not with antibiotics that did not target the cell wall. Similar synergistic killing was also demonstrated when KCl was utilized as the osmotic stressor. Conversely, osmolar equivalent concentrations of sucrose antagonized amoxicillin-mediated killing. Taken together, our results support a previously unrecognized interaction between amoxicillin resistance and osmotic stress in H. pylori. These findings have interesting implications for the effectiveness of antibiotic therapy for this pathogen. IMPORTANCE Rising antibiotic resistance rates in H. pylori are associated with increased rates of treatment failure. Understanding how stressors impact antibiotic resistance may shed light on the development of future treatment strategies. Previous studies found that mutations in PBP1 that conferred resistance to amoxicillin were also associated with a decrease in bacterial fitness. The current study demonstrated that osmotic stress can enhance beta lactam-mediated killing of H. pylori. The source of osmotic stress was found to be important for these interactions. Given that relatively little is known about how H. pylori responds to osmotic stress, these findings fill important knowledge gaps on this topic and provide interesting implications for the effectiveness of antibiotic therapy for this pathogen.

Use of the β-Glucan-Producing Lactic Acid Bacteria Strains Levilactobacillus brevis and Pediococcus claussenii for Sourdough Fermentation-Chemical Characterization and Chemopreventive Potential of In Situ-Enriched Wheat and Rye Sourdoughs and Breads.

The aim of the present study was to examine β-glucan production and the potential prebiotic and chemopreventive effects of wheat and rye sourdoughs and breads generated with wild-type and non-β-glucan-forming isogenic mutant strains of Levilactobacillus brevis and Pediococcus claussenii. Sourdough and bread samples were subjected to in vitro digestion and fermentation. Fermentation supernatants (FS) and pellets (FP) were analyzed (pH values, short-chain fatty acids (SCFA), ammonia, bacterial taxa) and the effects of FS on LT97 colon adenoma cell growth, viability, caspase-2 and -3 activity, genotoxic and antigenotoxic effects and on gene and protein expression of p21, cyclin D2, catalase and superoxide dismutase 2 (SOD2) were examined. Concentrations of SCFA were increased and concentrations of ammonia were partly reduced in the FS. The relative abundance of Bifidobacteriaceae was increased in all FPs. Treatment with FS reduced the growth and viability of LT97 cells and significantly increased caspase-2 and -3 activities without exhibiting genotoxic or antigenotoxic effects. The p21 mRNA and protein levels were increased while that of cyclin D2 was reduced. Catalase and SOD2 mRNA and protein expression were marginally induced. The presented results indicate a comparable chemopreventive potential of wheat and rye sourdoughs and breads without an additional effect of the formed β-glucan.

The Methyltransferase HemK Regulates the Virulence and Nutrient Utilization of the Phytopathogenic Bacterium Xanthomonas citri Subsp. citri.

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (Xcc), seriously affects fruit quality and yield, leading to significant economic losses around the world. Understanding the mechanism of Xcc virulence is important for the effective control of Xcc infection. In this report, we investigate the role of a protein named HemK in the regulation of the virulence traits of Xcc. The hemK gene was deleted in the Xcc jx-6 background, and the ΔhemK mutant phenotypically displayed significantly decreased motility, biofilm formation, extracellular enzymes, and polysaccharides production, as well as increased sensitivity to oxidative stress and high temperatures. In accordance with the role of HemK in the regulation of a variety of virulence-associated phenotypes, the deletion of hemK resulted in reduced virulence on citrus plants as well as a compromised hypersensitive response on a non-host plant, Nicotiana benthamiana. These results indicated that HemK is required for the virulence of Xcc. To characterize the regulatory effect of hemK deletion on gene expression, RNA sequencing analysis was conducted using the wild-type Xcc jx-6 strain and its isogenic ΔhemK mutant strain, grown in XVM2 medium. Comparative transcriptome analysis of these two strains revealed that hemK deletion specifically changed the expression of several virulence-related genes associated with the bacterial secretion system, chemotaxis, and quorum sensing, and the expression of various genes related to nutrient utilization including amino acid metabolism, carbohydrate metabolism, and energy metabolism. In conclusion, our results indicate that HemK plays an essential role in virulence, the regulation of virulence factor synthesis, and the nutrient utilization of Xcc.

New Insights on Gene by Environmental Effects of Drugs of Abuse in Animal Models Using GeneNetwork.

Gene-by-environment interactions are important for all facets of biology, especially behaviour. Families of isogenic strains of mice, such as the BXD strains, are excellently placed to study these interactions, as the same genome can be tested in multiple environments. BXD strains are recombinant inbred mouse strains derived from crossing two inbred strains—C57BL/6J and DBA/2J mice. Many reproducible genometypes can be leveraged, and old data can be reanalysed with new tools to produce novel insights. We obtained drug and behavioural phenotypes from Philip et al. Genes, Brain and Behaviour 2010, and reanalysed their data with new genotypes from sequencing, as well as new models (Genome-wide Efficient Mixed Model Association (GEMMA) and R/qtl2). We discovered QTLs on chromosomes 3, 5, 9, 11, and 14, not found in the original study. We reduced the candidate genes based on their ability to alter gene expression or protein function. Candidate genes included Slitrk6 and Cdk14. Slitrk6, in a Chromosome14 QTL for locomotion, was found to be part of a co-expression network involved in voluntary movement and associated with neuropsychiatric phenotypes. Cdk14, one of only three genes in a Chromosome5 QTL, is associated with handling induced convulsions after ethanol treatment, that is regulated by the anticonvulsant drug valproic acid. By using families of isogenic strains, we can reanalyse data to discover novel candidate genes involved in response to drugs of abuse.

Absence of Streptococcus pneumoniae Capsule Increases Bacterial Binding, Persistence, and Inflammation in Corneal Infection.

The role of the pneumococcal polysaccharide capsule is largely unclear for Streptococcus pneumoniae keratitis, an ocular inflammatory disease that develops as a result of bacterial infection of the cornea. In this study, capsule-deficient strains were compared to isogenic parent strains in their ability to adhere to human corneal epithelial cells. One isogenic pair was further used in topical ocular infection of mice to assess the contribution of the capsule to keratitis. The results showed that non-encapsulated pneumococci were significantly more adherent to cells, persisted in significantly higher numbers on mouse corneas in vivo, and caused significant increases in murine ocular IL9, IL10, IL12-p70, MIG, and MIP-1-gamma compared to encapsulated S. pneumoniae. These findings indicate that the bacterial capsule impedes virulence and the absence of capsule impacts inflammation following corneal infection.

Comparison of Isogenic Strains Shows No Evidence of Altered Nosocomial Transmission-Competency of Rough, GPL-Negative Mycobacterium abscessus Strains.

ABSTRACTMycobacterium abscessus is an emerging pathogen causing severe pulmonary infections. While environmental in origin, in the clinical setting M. abscessus often changes to a Rough phenotype associated with severe non-remitting infections. Clinical isolates baring mutations in glycopeptidolipid-synthesis genes, leading to the Rough phenotype, were suggested to have increase bacterial virulence while possibly showing reduced transmissibility on fomites. We set to determine whether an isolated glycopeptidolipid (GPL) defect affects transmissibility. We used transposon technology to create a fully isogenic Rough (GPL-defective) (Tn_4099c) and compare it to the isogenic parent strain (ATCC 19977). Survival on fomites was determined by spotting, drying, and retrieving the isolates at designated time points. This was repeated as a competition experiment using a mixture of differentially fluorescent M. abscessus 19977 (Smooth) and the Tn_4099c mutant (Rough). Survival ability in chlorhexidine solution (Septal Scrub Teva) was performed using a disinfectant killing-assay for mycobacteria. Despite significant bacterial killing in all assays, we found no survival advantage to either GPL-defected Rough or GPL-reserved Smooth morphotype—both on fomites and in chlorhexidine. Our findings suggest that while transmission fitness may be altered due to some within-host evolutionary changes, decreased transmissibility of clinical strains cannot be attributed to the GPL-synthesis defect alone. Further studies are needed to determine the effect of other mutations on the transmission potential of M. abscessus in the clinical setting.IMPORTANCE Mycobacterium abscessus is an emerging pathogen causing severe pulmonary infections. In the clinical setting, M. abscsssus undergoes molecular and genetic changes associated with increased virulence. Specifically, bacterial defects in glycopeptidolipid (GPL) synthesis, creating the “Rough” colony phenotype, have been associated with increased virulence, yet were also presumably observed to have decreased survival on fomites, leading to reduced transmissibility. We set to determine whether GPL-synthesis defects are indeed responsible for reduced transmissibility of clinical isolates. We compared fully isogenic GPL-disrupted versus GPL-preserved strains, and demonstrated no survival advantage for either strain on fomites. Additionally, neither isolate had a survival advantage in chlorhexidine, a widely used disinfectant in health care settings. Our findings suggest that reduced transmissibility of clinical isolates, should it be found, cannot be attributed to GPL-synthesis mutations. While clinical isolates may show changes in transmission potential, more studies are needed to investigate the mechanisms leading to these phenotypic changes.

CRISPR-Cas9 approach confirms Calcineurin-responsive zinc finger 1 (Crz1) transcription factor as a promising therapeutic target in echinocandin-resistant Candida glabrata

Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.

Aztreonam in combination with imipenem-relebactam against clinical and isogenic strains of serine and metallo-β-lactamase-producing enterobacterales

ObjectiveThe objective of this study was to evaluate the in vitro activity of aztreonam plus imipenem-relebactam against clinical and isogenic strains of Escherichia coli and Klebsiella pneumoniae co-harboring NDM and >1 serine β-lactamase. MethodsThirteen isolates were included: 4 clinical E. coli, 4 clinical K. pneumoniae, and 5 isogenic E. coli. Drugs were tested in time-kill analyses alone, in dual β-lactam combinations, and in triple drug combinations against all strains. ResultsAll isolates were resistant to imipenem and imipenem-relebactam, and 85% were aztreonam-resistant. Neither imipenem nor imipenem-relebactam was bactericidal alone while aztreonam was bactericidal against 54% of isolates. The combination of aztreonam+imipenem was bactericidal and synergistic against 7/13 and 10/13 isolates. The addition of relebactam to this combination resulted in synergy against all 11 aztreonam-resistant clinical isolates. ConclusionAztreonam plus imipenem-relebactam may be a viable treatment option for aztreonam-non-susceptible NDM and serine β-lactamase-producing E. coli and K. pneumoniae.

An acyclic phosphonate prodrug of HPMPC is effective against VZV in skin organ culture and mice

Varicella zoster virus (VZV) causes chicken pox and shingles and is prevalent worldwide. Acyclovir and penciclovir (and its prodrugs) are first-line treatments for VZV infections, but they are not highly potent against VZV and resistance may arise in immunocompromised people on long-term therapy. HPMPC (cidofovir) is active against VZV, but cidofovir is not approved for treating VZV diseases, is nephrotoxic, and is not orally bioavailable. Here, we present the synthesis and evaluation of USC-373, a phosphonate prodrug of HPMPC with activity against VZV and other DNA viruses. In cultured fibroblasts, it was potent against VZV Ellen laboratory strain and was not overtly toxic, with EC50 of 4 nM and CC50 of 0.20 μM, producing a selectivity index of 50. In ARPE-19 cells, USC-373 was effective against VZV-ORF57-Luc wild type strain and the acyclovir-resistant isogenic strain. In human skin organ culture, USC-373 formulated in cocoa butter and applied topically prevented VZV-ORF57-Luc spread without toxicity. In NuSkin mice with human skin xenografts, one daily dose of 3 mg/kg was effective by the subcutaneous route, and one daily dose of 10 mg/kg was effective by the oral route. Remarkably, a 10 mg/kg oral dose given every other day was also effective. USC-373 was well tolerated and mice did not lose weight or show signs of distress. The prodrug modifications of USC-373 increase the potency and oral bioavailability compared to its parent nucleoside analog, HPMPC.