Autochthonous faecal viral transfer (FVT) impacts the murine microbiome after antibiotic perturbation.

BACKGROUND: It has become increasingly accepted that establishing and maintaining a complex and diverse gut microbiota is fundamental to human health. There are growing efforts to identify means of modulating and influencing the microbiota, especially in individuals who have experienced a disruption in their native microbiota. Faecal microbiota transplantation (FMT) is one method that restores diversity to the microbiota of an individual by introducing microbes from a healthy donor. FMT introduces the total microbial load into the recipient, including the bacteria, archaea, yeasts, protists and viruses. In this study, we investigated whether an autochthonous faecal viral transfer (FVT), in the form of a sterile faecal filtrate, could impact the recovery of a bacteriome disrupted by antibiotic treatment. RESULTS: Following antibiotic disruption of the bacteriome, test mice received an FVT harvested prior to antibiotic treatment, while control mice received a heat- and nuclease-treated FVT. In both groups of mice, the perturbed microbiome reverted over time to one more similar to the pre-treatment one. However, the bacteriomes of mice that received an FVT, in which bacteriophages predominate, separated from those of the control mice as determined by principal co-ordinate analysis (PCoA). Moreover, analysis of the differentially abundant taxa indicated a closer resemblance to the pre-treatment bacteriome in the test mice that had received an FVT. Similarly, metagenomic sequencing of the virome confirmed that faecal bacteriophages of FVT and control mice differed over time in both abundance and diversity, with the phages constituting the FVT persisting in mice that received them. CONCLUSIONS: An autochthonous virome transfer reshaped the bacteriomes of mice post-antibiotic treatment such that they more closely resembled the pre-antibiotic microbiota profile compared to mice that received non-viable phages. Thus, FVT may have a role in addressing antibiotic-associated microbiota alterations and potentially prevent the establishment of post-antibiotic infection. Given that bacteriophages are biologically inert in the absence of their host bacteria, they could form a safe and effective alternative to whole microbiota transplants that could be delivered during/following perturbation of the gut flora.

Short-term consumption of a high-fat diet increases host susceptibility to Listeria monocytogenes infection.

BACKGROUND: A westernized diet comprising a high caloric intake from animal fats is known to influence the development of pathological inflammatory conditions. However, there has been relatively little focus upon the implications of such diets for the progression of infectious disease. Here, we investigated the influence of a high-fat (HF) diet upon parameters that influence Listeria monocytogenes infection in mice. RESULTS: We determined that short-term administration of a HF diet increases the number of goblet cells, a known binding site for the pathogen, in the gut and also induces profound changes to the microbiota and promotes a pro-inflammatory gene expression profile in the host. Host physiological changes were concordant with significantly increased susceptibility to oral L. monocytogenes infection in mice fed a HF diet relative to low fat (LF)- or chow-fed animals. Prior to Listeria infection, short-term consumption of HF diet elevated levels of Firmicutes including Coprococcus, Butyricicoccus, Turicibacter and Clostridium XIVa species. During active infection with L. monocytogenes, microbiota changes were further exaggerated but host inflammatory responses were significantly downregulated relative to Listeria-infected LF- or chow-fed groups, suggestive of a profound tempering of the host response influenced by infection in the context of a HF diet. The effects of diet were seen beyond the gut, as a HF diet also increased the sensitivity of mice to systemic infection and altered gene expression profiles in the liver. CONCLUSIONS: We adopted a systems approach to identify the effects of HF diet upon L. monocytogenes infection through analysis of host responses and microbiota changes (both pre- and post-infection). Overall, the results indicate that short-term consumption of a westernized diet has the capacity to significantly alter host susceptibility to L. monocytogenes infection concomitant with changes to the host physiological landscape. The findings suggest that diet should be a consideration when developing models that reflect human infectious disease.

A long and abundant non-coding RNA in Lactobacillus salivarius.

Lactobacillus salivarius, found in the intestinal microbiota of humans and animals, is studied as an example of the sub-dominant intestinal commensals that may impart benefits upon their host. Strains typically harbour at least one megaplasmid that encodes functions contributing to contingency metabolism and environmental adaptation. RNA sequencing (RNA-seq)transcriptomic analysis of L. salivarius strain UCC118 identified the presence of a novel unusually abundant long non-coding RNA (lncRNA) encoded by the megaplasmid, and which represented more than 75 % of the total RNA-seq reads after depletion of rRNA species. The expression level of this 520 nt lncRNA in L. salivarius UCC118 exceeded that of the 16S rRNA, it accumulated during growth, was very stable over time and was also expressed during intestinal transit in a mouse. This lncRNA sequence is specific to the L. salivarius species; however, among 45 L. salivarius genomes analysed, not all (only 34) harboured the sequence for the lncRNA. This lncRNA was produced in 27 tested L. salivarius strains, but at strain-specific expression levels. High-level lncRNA expression correlated with high megaplasmid copy number. Transcriptome analysis of a deletion mutant lacking this lncRNA identified altered expression levels of genes in a number of pathways, but a definitive function of this new lncRNA was not identified. This lncRNA presents distinctive and unique properties, and suggests potential basic and applied scientific developments of this phenomenon.

Development of a Click Beetle Luciferase Reporter System for Enhanced Bioluminescence Imaging of Listeria monocytogenes: Analysis in Cell Culture and Murine Infection Models.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is widely used as a model organism for the analysis of infection biology. In this context, there is a current need to develop improved reporters for enhanced bioluminescence imaging (BLI) of the pathogen in infection models. We have developed a click beetle red luciferase (CBR-luc) based vector (pPL2CBRopt) expressing codon optimized CBR-luc under the control of a highly expressed Listerial promoter (PHELP) for L. monocytogenes and have compared this to a lux-based system expressing bacterial luciferase for BLI of the pathogen using in vitro growth experiments and in vivo models. The CBR-luc plasmid stably integrates into the L. monocytogenes chromosome and can be used to label field isolates and laboratory strains of the pathogen. Growth experiments revealed that CBR-luc labeled L. monocytogenes emits a bright signal in exponential phase that is maintained during stationary phase. In contrast, lux-labeled bacteria produced a light signal that peaked during exponential phase and was significantly reduced during stationary phase. Light from CBR-luc labeled bacteria was more efficient than the signal from lux-labeled bacteria in penetrating an artificial tissue depth assay system. A cell invasion assay using C2Bbe1 cells and a systemic murine infection model revealed that CBR-luc is suited to BLI approaches and demonstrated enhanced sensitivity relative to lux in the context of Listeria infection models. Overall, we demonstrate that this novel CBR reporter system provides efficient, red-shifted light production relative to lux and may have significant applications in the analysis of L. monocytogenes pathogenesis.

Genome-Wide Search for Genes Required for Bifidobacterial Growth under Iron-Limitation.

Bacteria evolved over millennia in the presence of the vital micronutrient iron. Iron is involved in numerous processes within the cell and is essential for nearly all living organisms. The importance of iron to the survival of bacteria is obvious from the large variety of mechanisms by which iron may be acquired from the environment. Random mutagenesis and global gene expression profiling led to the identification of a number of genes, which are essential for Bifidobacterium breve UCC2003 survival under iron-restrictive conditions. These genes encode, among others, Fe-S cluster-associated proteins, a possible ferric iron reductase, a number of cell wall-associated proteins, and various DNA replication and repair proteins. In addition, our study identified several presumed iron uptake systems which were shown to be essential for B. breve UCC2003 growth under conditions of either ferric and/or ferrous iron chelation. Of these, two gene clusters encoding putative iron-uptake systems, bfeUO and sifABCDE, were further characterised, indicating that sifABCDE is involved in ferrous iron transport, while the bfeUO-encoded transport system imports both ferrous and ferric iron. Transcription studies showed that bfeUO and sifABCDE constitute two separate transcriptional units that are induced upon dipyridyl-mediated iron limitation. In the anaerobic gastrointestinal environment ferrous iron is presumed to be of most relevance, though a mutation in the sifABCDE cluster does not affect B. breve UCC2003's ability to colonise the gut of a murine model.

Listeria monocytogenes mutants defective in gallbladder replication represent safety-enhanced vaccine delivery platforms.

The Gram positive intracellular pathogen Listeria monocytogenes represents a promising vaccine or therapeutic DNA delivery vector that has been successfully administered to humans in clinical trials. However in generating Listeria mutants with therapeutic potential it is important to balance safety attenuation with efficacy. Here we show that L. monocytogenes mutants with a reduced capacity for murine gallbladder replication are capable of stimulating T cell responses in mice and protecting vaccinated animals from secondary challenge. Mutation of L. monocytogenes genes lmo2566 or lmo0598 resulted in significant attenuation in the murine model yet mutants retained a capacity for intracellular growth and stimulation of T cell responses against key Listeria epitopes (LLO91-99 and P60217-225). Importantly the mutants showed a reduced capacity for growth in the gallbladders of vaccinated mice as well as significantly reduced faecal shedding indicating that this approach generates live Listeria-based vector delivery systems with a reduced capacity for the spread of live genetically modified microorganisms into the natural environment.

Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in Listeria monocytogenes EGD-e and 10403S: roles in virulence and acid tolerance.

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains.

Exopolysaccharide-producing probiotic Lactobacilli reduce serum cholesterol and modify enteric microbiota in ApoE-deficient mice.

BACKGROUND: Probiotic bacteria have been associated with a reduction in cardiovascular disease risk, a leading cause of death and disability. OBJECTIVES: The aim of this study was to assess the impact of dietary administration of exopolysaccharide-producing probiotic Lactobacillus cultures on lipid metabolism and gut microbiota in apolipoprotein E (apoE)-deficient mice. METHODS: First, we examined lipid metabolism in response to dietary supplementation with recombinant ß-glucan-producing Lactobacillus paracasei National Food Biotechnology Centre (NFBC) 338 expressing the glycosyltransferase (Gtf) gene from Pediococcus parvulus 2.6 (GTF), and naturally exopolysaccharide-producing Lactobacillus mucosae Dairy Product Culture Collection (DPC) 6426 (DPC 6426) compared with the non-ß-glucan-producing isogenic control strain Lactobacillus paracasei NFBC 338 (PNZ) and placebo (15% wt:vol trehalose). Second, we examined the effects on the gut microbiota of dietary administration of DPC 6426 compared with placebo. Probiotic Lactobacillus strains at 1 × 10(9) colony-forming units/d per animal were administered to apoE(-/-) mice fed a high-fat (60% fat)/high-cholesterol (2% wt:wt) diet for 12 wk. At the end of the study, aortic plaque development and serum, liver, and fecal variables involved in lipid metabolism were analyzed, and culture-independent microbial analyses of cecal content were performed. RESULTS: Total cholesterol was reduced in serum (P < 0.001; ∼33-50%) and liver (P < 0.05; ∼30%) and serum triglyceride concentrations were reduced (P < 0.05; ∼15-25%) in mice supplemented with GTF or DPC 6426 compared with the PNZ or placebo group, respectively. In addition, dietary intervention with GTF led to increased amounts of fecal cholesterol excretion (P < 0.05) compared with all other groups. Compositional sequencing of the gut microbiota revealed a greater prevalence of Porphyromonadaceae (P = 0.001) and Prevotellaceae (P = 0.001) in the DPC 6426 group and lower proportions of Clostridiaceae (P < 0.05), Peptococcaceae (P < 0.001), and Staphylococcaceae (P < 0.01) compared with the placebo group. CONCLUSION: Ingestion of exopolysaccharide-producing lactobacilli resulted in seemingly favorable improvements in lipid metabolism, which were associated with changes in the gut microbiota of mice.

Autoinducer-2 plays a crucial role in gut colonization and probiotic functionality of Bifidobacterium breve UCC2003.

In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2), and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.

Two-tiered biological containment strategy for Lactococcus lactis-based vaccine or immunotherapy vectors.

The concept of biological containment was developed as a strategy to prevent environmental dissemination of engineered live vaccine or drug delivery vehicles. A mutation in the gene encoding thymidylate synthase (thyA), a key enzyme in the pyrimidine biosynthetic pathway, has previously been shown to limit growth of L. lactis vectors under restrictive conditions. We hypothesized that further mutations in the pyrimidine biosynthetic pathway might enhance the stability and safety of live L. lactis vectors. We show that a double mutation in the genes encoding ThyA and CTP synthase (PyrG) in L. lactis confers double auxotrophy for both thymidine and cytidine. However, the combination of two mutations failed to enhance the biological containment phenotype of the engineered strain. In the absence of thymine/thymidine, the thyA mutant exhibited a strong bactericidal phenotype. However, creation of the double mutant caused the loss of this phenotype, though survival in the mouse GI tract was enhanced. The implications for biological containment of live L. lactis based delivery vectors are discussed.

Bioavailability of the anti-clostridial bacteriocin thuricin CD in gastrointestinal tract.

Thuricin CD is a two component narrow spectrum bacteriocin comprising two peptides with targeted activity against Clostridium difficile. This study examined the bioavailability of thuricin with a view to developing it as an effective antimicrobial against intestinal infection. One of the peptides, Trn-ß, was found to be degraded by the gastric enzymes pepsin and α-chymotrypsin both in vitro and in vivo, whereas Trn-α was resistant to digestion by these enzymes and hence was detected in the intestinal porcine digesta following oral ingestion by pigs. In order to determine if spores of the producing organism Bacillus thuringiensis DPC 6431 could be used to deliver the bacteriocin to the gut, spores were fed to 30 mice (approx. 10(8)-2×10(8) per animal) and their germination, growth and production of thuricin in the gastrointestinal tract (GIT) of the animals was monitored. Almost 99 % of the spores delivered to the GIT were excreted in the first 24 h and neither Trn-α nor Trn-ß was detected in the gut or faecal samples of the test mice, indicating that ingestion of B. thuringiensis spores may not be a suitable vehicle for the delivery of thuricin CD. When thuricin CD was delivered rectally to mice (n = 40) and C. difficile shedding monitored at 1, 6, 12 and 24 h post-treatment, there was a >95 % (>1.5 log units) reduction of C. difficile 027 in the colon contents of infected mice (n = 10) 1 h post-treatment compared with the control group (n = 10; P<0.001). Furthermore, 6 h post-treatment there was a further 1.5 log reduction in C. difficile numbers (n = 10) relative to the control group (n = 10; P<0.05). These results would suggest that rectal administration of thuricin may be a promising mode of delivery of thuricin CD to the colon.

A mariner transposon-based signature-tagged mutagenesis system for the analysis of oral infection by Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listerosis a disease that manifests predominately as meningitis in the non-pregnant individual or infection of the fetus and spontaneous abortion in pregnant women. Common-source outbreaks of foodborne listeriosis are associated with significant morbidity and mortality. However, relatively little is known concerning the mechanisms that govern infection via the oral route. In order to aid functional genetic analysis of the gastrointestinal phase of infection we designed a novel signature-tagged mutagenesis (STM) system based upon the invasive L. monocytogenes 4b serotype H7858 strain. To overcome the limitations of gastrointestinal infection by L. monocytogenes in the mouse model we created a H7858 strain that is genetically optimised for oral infection in mice. Furthermore our STM system was based upon a mariner transposon to favour numerous and random transposition events throughout the L. monocytogenes genome. Use of the STM bank to investigate oral infection by L. monocytogenes identified 21 insertion mutants that demonstrated significantly reduced potential for infection in our model. The sites of transposon insertion included lmOh7858_0671 (encoding an internalin homologous to Lmo0610), lmOh7858_0898 (encoding a putative surface-expressed LPXTG protein homologous to Lmo0842), lmOh7858_2579 (encoding the HupDGC hemin transport system) and lmOh7858_0399 (encoding a putative fructose specific phosphotransferase system). We propose that this represents an optimised STM system for functional genetic analysis of foodborne/oral infection by L. monocytogenes.

In vivo assessment of growth and virulence gene expression during commensal and pathogenic lifestyles of luxABCDE-tagged Enterococcus faecalis strains in murine gastrointestinal and intravenous infection models.

Cytolysin and gelatinase are prominent pathogenicity determinants associated with highly virulent Enterococcus faecalis strains. In an effort to explore the expression profiles of these virulence traits in vivo, we have employed E. faecalis variants expressing the luxABCDE cassette under the control of either the P16S, cytolysin, or gelatinase promoter for infections of Galleria mellonella caterpillars and mice. Systemic infection of G. mellonella with bioluminescence-tagged E. faecalis MMH594 revealed temporal regulation of both gelatinase and cytolysin promoters and demonstrated that these traits were induced in response to the host environment. Gavage of mice pretreated perorally with antibiotics resulted in efficient colonization of the murine gastrointestinal tract (GIT) in a strain-dependent manner, where the commensal baby isolate EF62 was more persistent than the nosocomial isolate MMH594. A highly significant correlation (R(2) > 0.94) was found between bioluminescence and the CFU counts in mouse fecal samples. Both strains showed similar preferences for growth and persistence in the ileum, cecum, and colon. Cytolysin expression was uniform in these compartments of the intestinal lumen. In spite of high numbers (10(9) CFU/g of intestinal matter) in the ileum, cecum, and colon, no evidence of translocation or systemic infection could be observed. In the murine intravenous infection model, cytolysin expression was readily detected in the liver, kidneys, and bladder. At 72 h postinfection, the highest bacterial loads were found in the liver, kidneys, and spleen, with organ-specific expression levels of cytolysin ~400- and ~900-fold higher in the spleen and heart, respectively, than in the liver and kidneys. Taken together, this system based on the bioluminescence imaging technology is established as a new, powerful method to monitor the differential regulation of E. faecalis virulence determinants and to study the spatiotemporal course of infection in living animals in real time.

Virulence aspects of Listeria monocytogenes LO28 high pressure-resistant variants.

High pressure treatment is a novel food processing approach for reducing pathogens in foods and food ingredients. However, relatively little is known about the pathogenic potential of organisms that survive the treatment. Twelve previously isolated and characterized variants of Listeria monocytogenes LO28 obtained after a high pressure treatment were assessed for their virulence potential and antibiotic susceptibility. Ten variants showed attenuated virulence while two variants retained full virulence in a mouse model of infection. Seven of the attenuated variants demonstrated a reduction in virulence factor activity. Compared to the wild type, all variants exhibited similar or increased susceptibility to multiple antibiotics commonly used in listeriosis treatment.

In vivo activity of nisin A and nisin V against Listeria monocytogenes in mice.

BACKGROUND: Lantibiotics are post-translationally modified antimicrobial peptides, of which nisin A is the most extensively studied example. Bioengineering of nisin A has resulted in the generation of derivatives with increased in vitro potency against Gram-positive bacteria. Of these, nisin V (containing a Met21Val change) is noteworthy by virtue of exhibiting enhanced antimicrobial efficacy against a wide range of clinical and food-borne pathogens, including Listeria monocytogenes. However, this increased potency has not been tested in vivo. RESULTS: Here we address this issue by assessing the ability of nisin A and nisin V to control a bioluminescent strain of Listeria monocytogenes EGDe in a murine infection model.More specifically, Balb/c mice were infected via the intraperitoneal route at a dose of 1 × 10(5) cfu/animal and subsequently treated intraperitoneally with either nisin V, nisin A or a PBS control. Bioimaging of the mice was carried out on day 3 of the trial. Animals were then sacrificed and levels of infection were quantified in the liver and spleen. CONCLUSION: This analysis revealed that nisin V was more effective than Nisin A with respect to controlling infection and therefore merits further investigation with a view to potential chemotherapeutic applications.

A mutant in the Listeria monocytogenes Fur-regulated virulence locus (frvA) induces cellular immunity and confers protection against listeriosis in mice.

Listeria monocytogenes is a Gram-positive intracellular pathogen that is responsible for listeriosis, a potentially fatal, food-borne illness. Due to its cytoplasmic location during infection, this pathogen can mediate a long-lasting cellular immune response, which makes attenuated strains strong candidates for vaccine development. Recently, our group identified and characterized frvA (Fur-regulated virulence factor A), and deletion of this gene resulted in disruption of iron homeostasis and a strong attenuation in virulence. Despite significant attenuation in the mouse infection model, the frvA mutant was capable of intracellular growth in antigen-presenting cells. Indeed, mice immunized with L. monocytogenes ΔfrvA were able to effectively stimulate specific CD8(+) T cells to the listerial epitopes LLO(91-99) and P60(217-225) at levels comparable with L. monocytogenes strain EGDe. Most notably, mice immunized with ΔfrvA then subsequently challenged with the wild-type strain were completely protected from listerial infection. On the basis of these results, we advocate the use of ΔfrvA as a live attenuated listerial vaccine, and propose that this mutant may serve as a platform for the development of a future vaccine delivery vehicle.

Residual antibiotics disrupt meat fermentation and increase risk of infection.

UNLABELLED: Fermented sausages, although presumed safe for consumption, sometimes cause serious bacterial infections in humans that may be deadly. Not much is known about why and when this is the case. We tested the hypothesis that residual veterinary antibiotics in meat can disrupt the fermentation process, giving pathogenic bacteria a chance to survive and multiply. We found that six commercially available starter cultures were susceptible to commonly used antibiotics, namely, oxytetracycline, penicillin, and erythromycin. In meat, statutorily tolerable levels of oxytetracycline and erythromycin inhibited fermentation performance of three and five of the six starter cultures, respectively. In model sausages, the disruption of meat fermentation enhanced survival of the pathogens Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium compared to successful fermentations. Our work reveals an overlooked risk associated with the presence of veterinary drugs in meat. IMPORTANCE: Antibiotics have for a long time been used as growth promoters in farm animals, and while they are banned as such in Europe, their clinical use in farm animals still accounts for the majority of consumption. Here, we examined how acceptable levels of antibiotics in meat influence fermentation. Our results show that commonly used bacterial starter cultures are sensitive to residual antibiotics at or near statutorily tolerable levels, and as a result, processed sausages may indeed contain high levels of pathogens. Our findings provide a possible explanation for outbreaks and disease cases associated with consumption of fermented sausages and offer yet another argument for limiting the use of antimicrobials in farm animals.

H5N1 moratorium: Missing the point.

The recent moratorium on research using engineered H5N1 influenza viruses is a move which cannot achieve its aims as it ignores the prevalence of molecular biology.

HmgR, a key enzyme in the mevalonate pathway for isoprenoid biosynthesis, is essential for growth of Listeria monocytogenes EGDe.

Isoprenoids may be synthesized via one of two pathways, the classical mevalonate pathway or the alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. While the majority of bacteria utilize a single pathway for isoprenoid biosynthesis, Listeria monocytogenes is unusual in possessing the complete set of genes for both pathways. Here, we utilized new molecular tools to create precise gene deletions in selected genes encoding enzymes of both pathways, gcpE, lytB (encoding proteins in the MEP pathway) and hmgR (encoding a protein in the mevalonate pathway). We demonstrate that the hmgR gene can only be deleted when the growth medium is supplemented with exogenous mevalonate. Furthermore, full growth of the mutant in the absence of mevalonate was only possible when the intact hmgR gene was supplied in trans using an IPTG-inducible expression system. Murine competitive index assays performed via the oral and intraperitoneal routes of infection revealed that the mevalonate hmgR mutant could not be recovered from livers and spleens 3 days post-infection. We propose that HmgR in L. monocytogenes EGDe is involved in essential metabolic functions and that an intact MEP pathway is not capable of sustaining growth.