Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a.

Clostridium species are re-emerging as biotechnological workhorses for industrial acetone-butanol-ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25-100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.

Model-driven approach for the production of butyrate from CO2/H2 by a novel co-culture of C. autoethanogenum and C. beijerinckii.

One-carbon (C1) compounds are promising feedstocks for the sustainable production of commodity chemicals. CO2 is a particularly advantageous C1-feedstock since it is an unwanted industrial off-gas that can be converted into valuable products while reducing its atmospheric levels. Acetogens are microorganisms that can grow on CO2/H2 gas mixtures and syngas converting these substrates into ethanol and acetate. Co-cultivation of acetogens with other microbial species that can further process such products, can expand the variety of products to, for example, medium chain fatty acids (MCFA) and longer chain alcohols. Solventogens are microorganisms known to produce MCFA and alcohols via the acetone-butanol-ethanol (ABE) fermentation in which acetate is a key metabolite. Thus, co-cultivation of an acetogen and a solventogen in a consortium provides a potential platform to produce valuable chemicals from CO2. In this study, metabolic modeling was implemented to design a new co-culture of an acetogen and a solventogen to produce butyrate from CO2/H2 mixtures. The model-driven approach suggested the ability of the studied solventogenic species to grow on lactate/glycerol with acetate as co-substrate. This ability was confirmed experimentally by cultivation of Clostridium beijerinckii on these substrates in batch serum bottles and subsequently in pH-controlled bioreactors. Community modeling also suggested that a novel microbial consortium consisting of the acetogen Clostridium autoethanogenum, and the solventogen C. beijerinckii would be feasible and stable. On the basis of this prediction, a co-culture was experimentally established. C. autoethanogenum grew on CO2/H2 producing acetate and traces of ethanol. Acetate was in turn, consumed by C. beijerinckii together with lactate, producing butyrate. These results show that community modeling of metabolism is a valuable tool to guide the design of microbial consortia for the tailored production of chemicals from renewable resources.

Chemical Genetics Applied to Elucidate the Physiological Role of Stress-Signaling Molecules on the Wound-Induced Accumulation of Glucosinolates in Broccoli.

Wounding stress is an effective strategy to induce glucosinolate (GS) biosynthesis in broccoli. However, there is insufficient knowledge on the physiological and molecular mechanisms underlying this stress response. Herein, a chemical-genetic approach was applied to elucidate the role of jasmonic acid (JA), ethylene (ET), and reactive oxygen species (ROS) on the wound-induced biosynthesis of GS. Broccoli was processed into chops to induce wounding stress. Broccoli chops were treated with phenidone (PHEN) and diphenyleneiodonium chloride (DPI) as inhibitors of JA and ROS biosynthesis, respectively, whereas 1-methylcyclopropene (1-MCP) was applied as an inhibitor of ET action. Wounding stress induced the expression of genes related to the biosynthesis of indolic and aliphatic GS, which was correlated with the accumulation of GS and modulated by the inhibitors of signaling molecules applied. Results of gene expression analysis indicated that JA played a key role in the activation of most genes, followed by ROS. Furthermore, except for the CYP79B2 gene, PHEN and 1-MCP synergistically downregulated the expression of GS biosynthetic genes evaluated, showing that the interaction between JA and ET was fundamental to modulate GS biosynthesis. Results presented herein increased our knowledge of the physiological and molecular mechanisms governing the wound-induced biosynthesis of GS in broccoli.

Sporulation in solventogenic and acetogenic clostridia.

The Clostridium genus harbors compelling organisms for biotechnological production processes; while acetogenic clostridia can fix C1-compounds to produce acetate and ethanol, solventogenic clostridia can utilize a wide range of carbon sources to produce commercially valuable carboxylic acids, alcohols, and ketones by fermentation. Despite their potential, the conversion by these bacteria of carbohydrates or C1 compounds to alcohols is not cost-effective enough to result in economically viable processes. Engineering solventogenic clostridia by impairing sporulation is one of the investigated approaches to improve solvent productivity. Sporulation is a cell differentiation process triggered in bacteria in response to exposure to environmental stressors. The generated spores are metabolically inactive but resistant to harsh conditions (UV, chemicals, heat, oxygen). In Firmicutes, sporulation has been mainly studied in bacilli and pathogenic clostridia, and our knowledge of sporulation in solvent-producing or acetogenic clostridia is limited. Still, sporulation is an integral part of the cellular physiology of clostridia; thus, understanding the regulation of sporulation and its connection to solvent production may give clues to improve the performance of solventogenic clostridia. This review aims to provide an overview of the triggers, characteristics, and regulatory mechanism of sporulation in solventogenic clostridia. Those are further compared to the current knowledge on sporulation in the industrially relevant acetogenic clostridia. Finally, the potential applications of spores for process improvement are discussed.Key Points• The regulatory network governing sporulation initiation varies in solventogenic clostridia.• Media composition and cell density are the main triggers of sporulation.• Spores can be used to improve the fermentation process.

Transcriptomic and Phenotypic Analysis of a spoIIE Mutant in Clostridium beijerinckii.

SpoIIE is a phosphatase involved in the activation of the first sigma factor of the forespore, σ F , during sporulation. A ΔspoIIE mutant of Clostridium beijerinckii NCIMB 8052, previously generated by CRISPR-Cas9, did not sporulate but still produced granulose and solvents. Microscopy analysis also showed that the cells of the ΔspoIIE mutant are elongated with the presence of multiple septa. This observation suggests that in C. beijerinckii, SpoIIE is necessary for the completion of the sporulation process, as seen in Bacillus and Clostridium acetobutylicum. Moreover, when grown in reactors, the spoIIE mutant produced higher levels of solvents than the wild type strain. The impact of the spoIIE inactivation on gene transcription was assessed by comparative transcriptome analysis at three time points (4 h, 11 h and 23 h). Approximately 5% of the genes were differentially expressed in the mutant compared to the wild type strain at all time points. Out of those only 12% were known sporulation genes. As expected, the genes belonging to the regulon of the sporulation specific transcription factors (σ F , σ E , σ G , σ K ) were strongly down-regulated in the mutant. Inactivation of spoIIE also caused differential expression of genes involved in various cell processes at each time point. Moreover, at 23 h, genes involved in butanol formation and tolerance, as well as in cell motility, were up-regulated in the mutant. In contrast, several genes involved in cell wall composition, oxidative stress and amino acid transport were down-regulated. These results indicate an intricate interdependence of sporulation and stationary phase cellular events in C. beijerinckii.

Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii.

Recent developments in CRISPR technologies have opened new possibilities for improving genome editing tools dedicated to the Clostridium genus. In this study we adapted a two-plasmid tool based on this technology to enable scarless modification of the genome of two reference strains of Clostridium beijerinckii producing an Acetone/Butanol/Ethanol (ABE) or an Isopropanol/Butanol/Ethanol (IBE) mix of solvents. In the NCIMB 8052 ABE-producing strain, inactivation of the SpoIIE sporulation factor encoding gene resulted in sporulation-deficient mutants, and this phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. Furthermore, the fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome, resulting in mutants with endoglucanase activity. A similar two-plasmid approach was next used to edit the genome of the natural IBE-producing strain C. beijerinckii DSM 6423, which has never been genetically engineered before. Firstly, the catB gene conferring thiamphenicol resistance was deleted to make this strain compatible with our dual-plasmid editing system. As a proof of concept, our dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatB to remove the endogenous pNF2 plasmid, which led to a sharp increase of transformation efficiencies.

l-Rhamnose Metabolism in Clostridium beijerinckii Strain DSM 6423.

Macroalgae (or seaweeds) are considered potential biomass feedstocks for the production of renewable fuels and chemicals. Their sugar composition is different from that of lignocellulosic biomasses, and in green species, including Ulva lactuca, the major sugars are l-rhamnose and d-glucose. C. beijerinckii DSM 6423 utilized these sugars in a U. lactuca hydrolysate to produce acetic acid, butyric acid, isopropanol, butanol, and ethanol (IBE), and 1,2-propanediol. d-Glucose was almost completely consumed in diluted hydrolysates, while l-rhamnose or d-xylose was only partially utilized. In this study, the metabolism of l-rhamnose by C. beijerinckii DSM 6423 was investigated to improve its utilization from natural resources. Fermentations on d-glucose, l-rhamnose, and a mixture of d-glucose and l-rhamnose were performed. On l-rhamnose, the cultures showed low growth and sugar consumption and produced 1,2-propanediol, propionic acid, and n-propanol in addition to acetic and butyric acids, whereas on d-glucose, IBE was the major product. On a d-glucose-l-rhamnose mixture, both sugars were converted simultaneously and l-rhamnose consumption was higher, leading to high levels of 1,2-propanediol (78.4 mM), in addition to 59.4 mM butanol and 31.9 mM isopropanol. Genome and transcriptomics analysis of d-glucose- and l-rhamnose-grown cells revealed the presence and transcription of genes involved in l-rhamnose utilization and in bacterial microcompartment (BMC) formation. These data provide useful insights into the metabolic pathways involved in l-rhamnose utilization and the effects on the general metabolism (glycolysis, early sporulation, and stress response) induced by growth on l-rhamnose.IMPORTANCE A prerequisite for a successful biobased economy is the efficient conversion of biomass resources into useful products, such as biofuels and bulk and specialty chemicals. In contrast to other industrial microorganisms, natural solvent-producing clostridia utilize a wide range of sugars, including C5, C6, and deoxy-sugars, for production of long-chain alcohols (butanol and 2,3-butanediol), isopropanol, acetone, n-propanol, and organic acids. Butanol production by clostridia from first-generation sugars is already a commercial process, but for the expansion and diversification of the acetone, butanol, and ethanol (ABE)/IBE process to other substrates, more knowledge is needed on the regulation and physiology of fermentation of sugar mixtures. Green macroalgae, produced in aquaculture systems, harvested from the sea or from tides, can be processed into hydrolysates containing mixtures of d-glucose and l-rhamnose, which can be fermented. The knowledge generated in this study will contribute to the development of more efficient processes for macroalga fermentation and of mixed-sugar fermentation in general.

[Parotiditis in Chile: clinical and molecular characterization of two cases in a highly vaccinated population]. / Parotiditis en Chile: caracterización clínica y molecular de dos casos en una población altamente inmunizada.

Mumps virus usually produces a benign infection characterized by increased parotid volume which, prior to vaccination, mainly affected children and adolescents. After the introduction of measles, mumps and rubella (MMR) vaccine, mumps incidence decreased dramatically. This intervention also produced a change in its clinical presentation, moving to young adult patients, with an increased risk of complications. We report two clinical mumps cases in young adults with different clinical presentations. In both cases, serologic assays were assessed and, in one case, a polymerase chain reaction (PCR) was performed in order to confirm the diagnosis. The isolated virus was characterized and identifed as G genotype, the same genotype observed during outbreaks in United States and Europe, and different to the vaccinal strain. Mumps virus is currently circulating in Chile and it is important to be aware of possible outbreaks. Viral diagnosis can be difficult, particularly in populations with high vaccination coverage. Therefore, the access to etiologic study through PCR and serology becomes more relevant in order to optimize clinical management and secondary prevention measures.

Parotiditis en Chile: caracterización clínica y molecular de dos casos en una población altamente inmunizada / Parotiditis in Chile: clinical and molecular characterization of two cases in a highly vaccinated population

Resumen El virus de la parotiditis produce una infección benigna caracterizada por un aumento de volumen parotídeo que, antes de la introducción de la vacuna tres vírica, afectaba principalmente a niños y adolescentes. Luego de que esta vacuna se implementara en el Programa Nacional de Inmunizaciones, se produjo una notable disminución en su incidencia. Además, ocasionó un cambio en la edad y presentación clínica, siendo más frecuente en adultos jóvenes con mayor riesgo de complicaciones. Presentamos dos casos clínicos de parotiditis en adultos jóvenes confirmados por serología y en uno de ellos, por biología molecular. Se caracterizó el virus como del genotipo G, como el descrito en los brotes en E.U.A y Europa, diferente al virus contenido en la vacuna. El virus parotídeo sigue circulando en nuestro país y debemos mantenernos alerta ante eventuales brotes. Se hace relevante optimizar el diagnóstico etiológico por serología o técnicas de biología molecular con fines clínicos y epidemiológicos.

Mumps virus usually produces a benign infection characterized by increased parotid volume which, prior to vaccination, mainly affected children and adolescents. After the introduction of measles, mumps and rubella (MMR) vaccine, mumps incidence decreased dramatically. This intervention also produced a change in its clinical presentation, moving to young adult patients, with an increased risk of complications. We report two clinical mumps cases in young adults with different clinical presentations. In both cases, serologic assays were assessed and, in one case, a polymerase chain reaction (PCR) was performed in order to confirm the diagnosis. The isolated virus was characterized and identifed as G genotype, the same genotype observed during outbreaks in United States and Europe, and different to the vaccinal strain. Mumps virus is currently circulating in Chile and it is important to be aware of possible outbreaks. Viral diagnosis can be difficult, particularly in populations with high vaccination coverage. Therefore, the access to etiologic study through PCR and serology becomes more relevant in order to optimize clinical management and secondary prevention measures.

Evaluation of monoclonal antibodies that detect conserved proteins from Respiratory Syncytial Virus, Metapneumovirus and Adenovirus in human samples.

Human Respiratory Syncytial Virus (hRSV), human Metapneumovirus (hMPV) and Adenovirus (ADV), are three of the most prevalent viruses responsible for pneumonia and bronchiolitis in children and elderly worldwide, accounting for a high number of hospitalizations annually. Diagnosis of these viruses is required to take clinical actions that allow an appropriate patient management. Thereby, new strategies to design fast diagnostic methods are highly required. In the present work, six monoclonal antibodies (mAbs, two for each virus) specific for conserved proteins from hRSV, hMPV and ADV were generated and evaluated through different immunological techniques, based on detection of purified protein, viral particles and human samples. In vitro evaluation of these antibodies showed higher specificity and sensitivity than commercial antibodies tested in this study. These antibodies were used to design a sandwich ELISA tests that allowed the detection of hRSV, hMPV, and ADV in human nasopharyngeal swabs. We observed that hRSV and ADV were detected with sensitivity and specificity equivalent to a current Direct Fluorescence Assay (DFA) methodology. However, hMPV was detected with more sensitivity than DFA. Our data suggest that these new mAbs can efficiently identify infected samples and discriminate from patients infected with other respiratory pathogens.

A two-plasmid inducible CRISPR/Cas9 genome editing tool for Clostridium acetobutylicum.

CRISPR/Cas-based genetic engineering has revolutionised molecular biology in both eukaryotes and prokaryotes. Several tools dedicated to the genomic transformation of the Clostridium genus of Gram-positive bacteria have been described in the literature; however, the integration of large DNA fragments still remains relatively limited. In this study, a CRISPR/Cas9 genome editing tool using a two-plasmid strategy was developed for the solventogenic strain Clostridium acetobutylicum ATCC 824. Codon-optimised cas9 from Streptococcus pyogenes was placed under the control of an anhydrotetracycline-inducible promoter on one plasmid, while the gRNA expression cassettes and editing templates were located on a second plasmid. Through the sequential introduction of these vectors into the cell, we achieved highly accurate genome modifications, including nucleotide substitution, gene deletion and cassette insertion up to 3.6kb. To demonstrate its potential, this genome editing tool was used to generate a marker-free mutant of ATCC 824 that produced an isopropanol-butanol-ethanol mixture. Whole-genome sequencing confirmed that no off-target modifications were present in the mutants. Such a tool is a prerequisite for efficient metabolic engineering in this solventogenic strain and provides an alternative editing strategy that might be applicable to other Clostridium strains.

Biorefinery of the green seaweed Ulva lactuca to produce animal feed, chemicals and biofuels.

The growing world population demands an increase in animal protein production. Seaweed may be a valuable source of protein for animal feed. However, a biorefinery approach aimed at cascading valorisation of both protein and non-protein seaweed constituents is required to realise an economically feasible value chain. In this study, such a biorefinery approach is presented for the green seaweed Ulva lactuca containing 225 g protein (N × 4.6) kg-1 dry matter (DM). The sugars in the biomass were solubilised by hot water treatment followed by enzymatic hydrolysis and centrifugation resulting in a sugar-rich hydrolysate (38.8 g L-1 sugars) containing glucose, rhamnose and xylose, and a protein-enriched (343 g kg-1 in DM) extracted fraction. This extracted fraction was characterised for use in animal feed, as compared to U. lactuca biomass. Based on the content of essential amino acids and the in vitro N (85 %) and organic matter (90 %) digestibility, the extracted fraction seems a promising protein source in diets for monogastric animals with improved characteristics as compared to the intact U. lactuca. The gas production test indicated a moderate rumen fermentation of U. lactuca and the extracted fraction, about similar to that of alfalfa. Reduction of the high content of minerals and trace elements may be required to allow a high inclusion level of U. lactuca products in animal diets. The hydrolysate was used successfully for the production of acetone, butanol, ethanol and 1,2-propanediol by clostridial fermentation, and the rhamnose fermentation pattern was studied.

Cost-Effectiveness Analysis of Different Testing Strategies that Use Antibody Levels to Detect Chronic Hepatitis C in Blood Donors.

AIM: We conducted a cost-effectiveness analysis of seven hepatitis C virus (HCV) testing strategies in blood donors. METHODS: Three of the seven strategies were based on HCV diagnosis and reporting guidelines in Mexico and four were from previous and current recommendations outlined by the CDC. The strategies that were evaluated determine antibody levels according to the signal-to-cut-off (S/CO) ratio and use reflex Immunoblot (IMB) or HCV RNA tests to confirm true positive (TP) cases of chronic HCV infection. Costs were calculated from the perspective of the Mexican Institute of Social Security (IMSS). A decision tree model was developed to estimate the expected number of true positive cases and costs for the base-case scenarios and for the sensitivity analyses. RESULTS: Base-case findings indicate an extended dominance of the CDC-USA2 and CDC-USA4 options by the IMSS Mexico3 and IMSS-Mexico1 alternatives. The probabilistic sensitivity analyses results suggest that for a willingness-to-pay (WTP) range of $0-9,000 USD the IMSS-Mexico1 strategy is the most cost-effective of all strategies ($5,000 USD per TP). The IMSS-Mexico3, IMSS-Mexico2, and CDC-USA3 strategies are also cost-effective strategies that cost between $7,800 and $8,800 USD per TP case detected. The CDC-USA1 strategy was very expensive and not cost-effective. CONCLUSIONS: HCV antibody testing strategies based on the classification of two or three levels of the S/CO are cost-effective procedures to identify patients who require reflex IMB or HCV RNA testing to confirm chronic HCV infection.

Cetuximab-oxaliplatin-liposomes for epidermal growth factor receptor targeted chemotherapy of colorectal cancer.

Oxaliplatin (L-OH), a platinum derivative with good tolerability is currently combined with Cetuximab (CTX), a monoclonal antibody (mAb), for the treatment of certain (wild-type KRAS) metastatic colorectal cancer (CRC) expressing epidermal growth factor receptor (EGFR). Improvement of L-OH pharmacokinetics (PK) can be provided by its encapsulation into liposomes, allowing a more selective accumulation and delivery to the tumor. Here, we aim to associate both agents in a novel liposomal targeted therapy by linking CTX to the drug-loaded liposomes. These EGFR-targeted liposomes potentially combine the therapeutic activity and selectivity of CTX with tumor-cell delivery of L-OH in a single therapeutic approach. L-OH liposomes carrying whole CTX or CTX-Fab' fragments on their surface were designed and characterized. Their functionality was tested in vitro using four human CRC cell lines, expressing different levels of EGFR to investigate the role of CTX-EGFR interactions in the cellular binding and uptake of the nanocarriers and encapsulated drug. Next, those formulations were evaluated in vivo in a colorectal cancer xenograft model with regard to tumor drug accumulation, toxicity and therapeutic activity. In EGFR-overexpressing cell lines, intracellular drug delivery by targeted liposomes increased with receptor density reaching up to 3-fold higher levels than with non-targeted liposomes. Receptor specific uptake was demonstrated by competition with free CTX, which reduced internalization to levels similar to non-targeted liposomes. In a CRC xenograft model, drug delivery was strongly enhanced upon treatment with targeted formulations. Liposomes conjugated with monovalent CTX-Fab' fragments showed superior drug accumulation in tumor tissue (2916.0±507.84ng/g) compared to CTX liposomes (1546.02±362.41ng/g) or non-targeted liposomes (891.06±155.1ng/g). Concomitantly, CTX-Fab' targeted L-OH liposomes outperformed CTX-liposomes, which on its turn was still more efficacious than non-targeted liposomes and free drug treatment in CRC bearing mice. These results show that site-directed conjugation of monovalent CTX-Fab' provides targeted L-OH liposomes that display an increased tumor drug delivery and efficacy over a formulation with CTX and non-targeted liposomes.

[Accuracy and diagnostic utility of IgM in Bartonella henselae infections]. / Exactitud y utilidad diagnóstica de la IgM en infecciones por Bartonella henselae.

INTRODUCTION: Laboratory diagnosis of cat scratch disease (CSD) is based on the determination of specific antibodies anti-Bartonella henselae by different techniques. The CDC recommends IgG by immunofluorescent assay (IFA) as the gold standard. OBJECTIVE: To determine the accuracy and diagnostic utility of anti-B.henselae IgM by IFA for CSD. MATERIAL AND METHODS: Anti-B. henselae IgG was determined in serum of 108 patients with CSD suspicion; in addition, specific IgM was determined separately and blindly by two thoroughly trained laboratory professionals. We calculated sensitivity (S), specificity (Sp), predictive values both positive (PPV) and negative (NPV), and likelihood ratio (LR) for IgM positive (LR +) and negative (LR-). RESULTS: In 37 patients with positive anti-B.henselae IgG, IgM was positive in 16 and negative in 21; in 71 patients with negative IgG, IgM was negative in 69 and positive in 2. Therefore, IgM showed S 43%, E 97%, PPV 88%, NPV 77%, LR (+) 15 and LR (-) 0.58. CONCLUSIONS: The results show that a positive IgM supports, but a negative one does not rule out a B. henselae infection. Therefore, IgG should be still considered as the gold standard for the diagnosis of CSD.

"In situ" removal of isopropanol, butanol and ethanol from fermentation broth by gas stripping.

In this study, the removal of IBE from aqueous solutions by gas stripping has been characterized. The effect of one or more components in the solution on the kinetics of the separation has been studied, both at 37°C and at 70°C. Gas stripping has been applied to batch, repeated batch and continuous cultures of Clostridium beijerinckii grown on a glucose/xylose mixed sugar substrate mimicking lignocellulosic hydrolysates, with the aim of finding optimal conditions for a stable IBE-producing culture with high productivity. An innovative repeated-batch process has been demonstrated in which the gas-stripping is performed at 70°C, resulting in a prolonged stable IBE culture.

Exactitud y utilidad diagnóstica de la IgM en infecciones por Bartonella henselae / Accuracy and diagnostic utility of IgM in Bartonella henselae infections

Introduction: Laboratory diagnosis of cat scratch disease (CSD) is based on the determination of specific antibodies anti-Bartonella henselae by different techniques. The CDC recommends IgG by immunofluorescent assay (IFA) as the gold standard. Objective: To determine the accuracy and diagnostic utility of anti-B.henselae IgM by IFA for CSD. Material and Methods: Anti-B. henselae IgG was determined in serum of 108 patients with CSD suspicion; in addition, specific IgM was determined separately and blindly by two thoroughly trained laboratory professionals. We calculated sensitivity (S), specificity (Sp), predictive values both positive (PPV) and negative (NPV), and likelihood ratio (LR) for IgM positive (LR +) and negative (LR-). Results: In 37 patients with positive anti-B.henselae IgG, IgM was positive in 16 and negative in 21; in 71 patients with negative IgG, IgM was negative in 69 and positive in 2. Therefore, IgM showed S 43%, E 97%, PPV 88%, NPV 77%, LR (+) 15 and LR (-) 0.58. Conclusions: The results show that a positive IgM supports, but a negative one does not rule out a B. henselae infection. Therefore, IgG should be still considered as the gold standard for the diagnosis of CSD.

Introducción: El diagnóstico de laboratorio de la enfermedad por arañazo de gato (EAG) se basa en la determinación de anticuerpos específicos anti-Bartonella henselae por distintas técnicas. El CDC de E.U.A. recomienda como estándar de oro la IgG mediante inmunofluorescencia (IF). Objetivo: Determinar la exactitud y utilidad diagnóstica de la IgM anti-B. henselae por IF para EAG. Material y Método: En suero de 108 pacientes a quienes se realizó IgG anti-B. henselae por sospecha de EAG, se determinó la presencia de IgM específica, en forma separada y ciega por dos profesionales de laboratorio ampliamente entrenados. Se calculó sensibilidad (S), especificidad (E), valores predictores positivo (VPP) y negativo (VPN) y likelihood ratio (LR) para una IgM positiva (LR+) y negativa (LR-). Resultados: En 37 pacientes con IgG anti-B. henselae positiva, la IgM fue positiva en 16 y negativa en 11; en 71 pacientes con IgG negativa, la IgM fue negativa en 69 y positiva en 2. Por consiguiente, la IgM presentó S 43%, E 97%, VPP 88%, VPN 77%, LR(+) 15 y LR(-) 0,58. Conclusiones: Los resultados sugieren que una IgM positiva apoya el diagnóstico de EAG, pero una negativa no permite descartarlo. Por tanto, la IgG debe seguir considerándose como el estándar de oro para el diagnóstico de infecciones por B. henselae.

Production of acetone, butanol, and ethanol from biomass of the green seaweed Ulva lactuca.

Green seaweed Ulva lactuca harvested from the North Sea near Zeeland (The Netherlands) was characterized as feedstock for acetone, ethanol and ethanol fermentation. Solubilization of over 90% of sugars was achieved by hot-water treatment followed by hydrolysis using commercial cellulases. A hydrolysate was used for the production of acetone, butanol and ethanol (ABE) by Clostridium acetobutylicum and Clostridium beijerinckii. Hydrolysate-based media were fermentable without nutrient supplementation. C. beijerinckii utilized all sugars in the hydrolysate and produced ABE at high yields (0.35 g ABE/g sugar consumed), while C. acetobutylicum produced mostly organic acids (acetic and butyric acids). These results demonstrate the great potential of U. lactuca as feedstock for fermentation. Interestingly, in control cultures of C. beijerinckii on rhamnose and glucose, 1,2 propanediol was the main fermentation product (9.7 g/L).

Simultaneous production of isopropanol, butanol, ethanol and 2,3-butanediol by Clostridium acetobutylicum ATCC 824 engineered strains.

Isopropanol represents a widely-used commercial alcohol which is currently produced from petroleum. In nature, isopropanol is excreted by some strains of Clostridium beijerinckii, simultaneously with butanol and ethanol during the isopropanol butanol ethanol (IBE) fermentation. In order to increase isopropanol production, the gene encoding the secondary-alcohol dehydrogenase enzyme from C. beijerinckii NRRL B593 (adh) which catalyzes the reduction of acetone to isopropanol, was cloned into the acetone, butanol and ethanol (ABE)-producing strain C. acetobutylicum ATCC 824. The transformants showed high capacity for conversion of acetone into isopropanol (> 95%). To increase isopropanol production levels in ATCC 824, polycistronic transcription units containing, in addition to the adh gene, homologous genes of the acetoacetate decarboxylase (adc), and/or the acetoacetyl-CoA:acetate/butyrate:CoA transferase subunits A and B (ctfA and ctfB) were constructed and introduced into the wild-type strain. Combined overexpression of the ctfA and ctfB genes resulted in enhanced solvent production. In non-pH-controlled batch cultures, the total solvents excreted by the transformant overexpressing the adh, ctfA, ctfB and adc genes were 24.4 g/L IBE (including 8.8 g/L isopropanol), while the control strain harbouring an empty plasmid produced only 20.2 g/L ABE (including 7.6 g/L acetone). The overexpression of the adc gene had limited effect on IBE production. Interestingly, all transformants with the adh gene converted acetoin (a minor fermentation product) into 2,3-butanediol, highlighting the wide metabolic versatility of solvent-producing Clostridia.

Disruption of the acetate kinase (ack) gene of Clostridium acetobutylicum results in delayed acetate production.

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.