Identification of a CTX-M-255 ß-lactamase containing a G239S substitution selectively conferring resistance to penicillin/ß-lactamase inhibitor combinations.

OBJECTIVES: An Escherichia coli isolate, WGS1363, showed resistance to piperacillin/tazobactam but susceptibility to cephalosporins and contained a previously unrecognized ß-lactamase, CTX-M-255, as the only acquired ß-lactamase. CTX-M-255 was identical to CTX-M-27 except for a G239S substitution. Here, we characterize the hydrolytic spectrum of CTX-M-255 and a previously reported ß-lactamase, CTX-M-178, also containing a G239S substitution and compare it to their respective parental enzymes, CTX-M-27 and CTX-M-15. METHODS: All ß-lactamase genes were expressed in E. coli TOP10 and MICs to representative ß-lactam-antibiotics were determined. Furthermore, blaCTX-M-15,  blaCTX-M-27, blaCTX-M-178 and blaCTX-M-255 with C-terminal His-tag fusions were affinity purified for enzyme kinetic assays determining Michaelis-Menten kinetic parameters against representative ß-lactam-antibiotics and IC50s of clavulanate, sulbactam, tazobactam and avibactam. RESULTS: TOP10-transformants expressing blaCTX-M-178 and blaCTX-M-255 showed resistance to penicillin/ß-lactamase combinations and susceptibility to cephalothin and cefotaxime in contrast to transformants expressing blaCTX-M-15 and blaCTX-M-27. Determination of enzyme kinetic parameters showed that CTX-M-178 and CTX-M-255 both lacked hydrolytic activity against cephalosporins and showed impaired hydrolytic efficiency against penicillin antibiotics compared to their parental enzymes. Both enzymes appeared more active against piperacillin compared to benzylpenicillin and ampicillin. Compared to their parental enzymes, IC50s of ß-lactamase-inhibitors were increased more than 1000-fold for CTX-M-178 and CTX-M-255. CONCLUSIONS: CTX-M-178 and CTX-M-255, both containing a G239S substitution, conferred resistance to piperacillin/tazobactam and may be characterized as inhibitor-resistant CTX-M ß-lactamases. Inhibitor resistance was accompanied by loss of activity against cephalosporins and monobactams. These findings add to the necessary knowledge base for predicting antibiotic susceptibility from genotypic data.

The global transcriptomes of Salmonella enterica serovars Gallinarum, Dublin and Enteritidis in the avian host.

Salmonella enterica serovar Gallinarum causes Fowl Typhoid in poultry, and it is host specific to avian species. The reasons why S. Gallinarum is restricted to avians, and at the same time predominately cause systemic infections in these hosts, are unknown. In the current study, we developed a surgical approach to study gene expression inside the peritoneal cavity of hens to shed light on this. Strains of the host specific S. Gallinarum, the cattle-adapted S. Dublin and the broad host range serovar, S. Enteritidis, were enclosed in semi-permeable tubes and surgically placed for 4 h in the peritoneal cavity of hens and for control in a minimal medium at 41.2 °C. Global gene-expression under these conditions was compared between serovars using tiled-micro arrays with probes representing the genome of S. Typhimurium, S. Dublin and S. Gallinarum. Among other genes, genes of SPI-13, SPI-14 and the macrophage survival gene mig-14 were specifically up-regulated in the host specific serovar, S. Gallinarum, and further studies into the role of these genes in host specific infection are highly indicated. Analysis of pathways and GO-terms, which were enriched in the host specific S. Gallinarum without being enriched in the two other serovars indicated that host specificity was characterized by a metabolic fine-tuning as well as unique expression of virulence associated pathways. The cattle adapted serovar S. Dublin differed from the two other serovars by a lack of up-regulation of genes encoded in the virulence associated pathogenicity island 2, and this may explain the inability of this serovar to cause disease in poultry.

Global transcriptomic response of the AI-3 isomers 3,5-DPO and 3,6-DPO in Salmonella Typhimurium.

Bacterial intercellular signaling mediated by small molecules, also called autoinducers (AIs), enables synchronized behavior in response to environmental conditions, and in many bacterial pathogens, intercellular signaling controls virulence gene expression. However, in the intestinal pathogen Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), although three signals, named AI-1, AI-2 and AI-3, have been described, their roles in virulence remain elusive. AI-3 is the 3,6- isomer of a previously described Vibrio cholerae signaling molecule; 3,5-dimethylpyrazin-2-ol (3,5-DPO). To elucidate the role of AI-3/DPO in S. Typhimurium, we have mapped the global transcriptomic responses to 3,5- and 3,6-DPO isomers in S. Typhimurium. Our studies showed that DPO affects expression of almost 8% of all genes. Specifically, expression of several genes involved in gut-colonization respond to DPO. Interestingly, most of the affected genes are similarly regulated by 3,5-DPO and 3,6-DPO, respectively, indicating that the two isomers have overlapping roles in S. Typhimurium.

Resistance to piperacillin/tazobactam in Escherichia coli resulting from extensive IS26-associated gene amplification of blaTEM-1.

BACKGROUND: bla TEM-1 encodes a narrow-spectrum ß-lactamase that is inhibited by ß-lactamase inhibitors and commonly present in Escherichia coli. Hyperproduction of blaTEM-1 may cause resistance to penicillin/ß-lactamase inhibitor (P/BLI) combinations. OBJECTIVES: To characterize EC78, an E. coli bloodstream isolate, resistant to P/BLI combinations, which contains extensive amplification of blaTEM-1 within the chromosome. METHODS: EC78 was sequenced using Illumina and Oxford Nanopore Technology (ONT) methodology. Configuration of blaTEM-1 amplification was probed using PCR. Expression of blaTEM-1 mRNA was determined using quantitative PCR and ß-lactamase activity was determined spectrophotometrically in a nitrocefin conversion assay. Growth rate was assessed to determine fitness and stability of the gene amplification was assessed by passage in the absence of antibiotics. RESULTS: Illumina sequencing of EC78 identified blaTEM-1B as the only acquired ß-lactamase preceded by the WT P3 promoter and present at a copy number of 182.6 with blaTEM-1B bracketed by IS26 elements. The chromosomal location of the IS26-blaTEM-1B amplification was confirmed by ONT sequencing. Hyperproduction of blaTEM-1 was confirmed by increased transcription of blaTEM-1 and ß-lactamase activity and associated with a significant fitness cost; however, the array was maintained at a relatively high copy number for 150 generations. PCR screening for blaTEM amplification of isolates resistant to P/BLI combinations identified an additional strain containing an IS26-associated amplification of a blaTEM gene. CONCLUSIONS: IS26-associated amplification of blaTEM can cause resistance to P/BLI combinations. This adaptive mechanism of resistance may be overlooked if simple methods of genotypic prediction (e.g. gene presence/absence) are used to predict antimicrobial susceptibility from sequencing data.

Putrescine biosynthesis and export genes are essential for normal growth of avian pathogenic Escherichia coli.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T). RESULTS: Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed. CONCLUSIONS: Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.

The In Vitro Redundant Enzymes PurN and PurT Are Both Essential for Systemic Infection of Mice in Salmonella enterica Serovar Typhimurium.

Metabolic enzymes show a high degree of redundancy, and for that reason they are generally ignored in searches for novel targets for anti-infective substances. The enzymes PurN and PurT are redundant in vitro in Salmonella enterica serovar Typhimurium, in which they perform the third step of purine synthesis. Surprisingly, the results of the current study demonstrated that single-gene deletions of each of the genes encoding these enzymes caused attenuation (competitive infection indexes [CI] of <0.03) in mouse infections. While the ΔpurT mutant multiplied as fast as the wild-type strain in cultured J774A.1 macrophages, net multiplication of the ΔpurN mutant was reduced approximately 50% in 20 h. The attenuation of the ΔpurT mutant was abolished by simultaneous removal of the enzyme PurU, responsible for the formation of formate, indicating that the attenuation was related to formate accumulation or wasteful consumption of formyl tetrahydrofolate by PurU. In the process of further characterization, we disclosed that the glycine cleavage system (GCV) was the most important for formation of C1 units in vivo (CI = 0.03 ± 0.03). In contrast, GlyA was the only important enzyme for the formation of C1 units in vitro The results with the ΔgcvT mutant further revealed that formation of serine by SerA and further conversion of serine into C1 units and glycine by GlyA were not sufficient to ensure C1 formation in S Typhimurium in vivo The results of the present study call for reinvestigations of the concept of metabolic redundancy in S Typhimurium in vivo.

Salmonella Typhimurium undergoes distinct genetic adaption during chronic infections of mice.

BACKGROUND: Typhoid fever caused by Salmonella enterica serovar Typhi (S. Typhi) is a severe systemic human disease and endemic in regions of the world with poor drinking water quality and sewage treatment facilities. A significant number of patients become asymptomatic life-long carriers of S. Typhi and serve as the reservoir for the disease. The specific mechanisms and adaptive strategies enabling S. Typhi to survive inside the host for extended periods are incompletely understood. Yet, elucidation of these processes is of major importance for improvement of therapeutic strategies. In the current study genetic adaptation during experimental chronic S. Typhimurium infections of mice, an established model of chronic typhoid fever, was probed as an approach for studying the molecular mechanisms of host-adaptation during long-term host-association. RESULTS: Individually sequence-tagged wild type strains of S. Typhimurium 4/74 were used to establish chronic infections of 129X1/SvJ mice. Over the course of infections, S. Typhimurium bacteria were isolated from feces and from livers and spleens upon termination of the experiment. In all samples dominant clones were identified and select clones were subjected to whole genome sequencing. Dominant clones isolated from either systemic organs or fecal samples exhibited distinct single nucleotide polymorphisms (SNPs). One mouse appeared to have distinct adapted clones in the spleen and liver, respectively. Three mice were colonized in the intestines by the same clone containing the same non-synonymous SNP in a transcriptional regulator, kdgR, of metabolic genes. This likely indicates transmission of this clone between mice. The mutation was tracked to have occurred prior to 2 weeks post infection in one of the three mice and had subsequently been transmitted to the other two mice. Re-infection with this clone confirmed that it is superior to the wild type for intestinal colonization. CONCLUSIONS: During 4 to 6 weeks of chronic infections, S. Typhimurium acquired distinct SNPs in known regulators of metabolic and virulence genes. One SNP, the kdgR-SNP was confirmed to confer selective advantage during chronic infections and constitute a true patho-adaptive mutation. Together, the results provide evidence for rapid genetic adaptation to the host of S. Typhimurium and validate experimental evolution in the context of host infection as a strategy for elucidating pathogen host interactions at the molecular level.

Multiple roles of putrescine and spermidine in stress resistance and virulence of Salmonella enterica serovar Typhimurium.

Polyamines (putrescine and spermidine) are small-cationic amines ubiquitous in nature and present in most living cells. In recent years they have been linked to virulence of several human pathogens including Shigella spp and Salmonella enterica serovar Typhimurium (S. Typhimurium). Central to S. Typhimurium virulence is the ability to survive and replicate inside macrophages and resisting the antimicrobial attacks in the form of oxidative and nitrosative stress elicited from these cells. In the present study, we have investigated the role of polyamines in intracellular survival and systemic infections of mice. Using a S. Typhimurium mutant defective for putrescine and spermidine biosynthesis, we show that polyamines are essential for coping with reactive nitrogen species, possibly linking polyamines to increased intracellular stress resistance. However, using a mouse model defective for nitric oxide production, we find that polyamines are required for systemic infections independently of host-produced reactive nitrogen species. To distinguish between the physiological roles of putrescine and spermidine, we constructed a strain deficient for spermidine biosynthesis and uptake, but with retained ability to produce and import putrescine. Interestingly, in this mutant we observe a strong attenuation of virulence during infection of mice proficient and deficient for nitric oxide production suggesting that spermidine, specifically, is essential for virulence of S. Typhimurium.

CTX-M-1 ß-lactamase expression in Escherichia coli is dependent on cefotaxime concentration, growth phase and gene location.

OBJECTIVES: Knowledge about the regulatory mechanisms of CTX-M ß-lactamase-encoding genes in Escherichia coli is limited. The objectives of this study were to determine the growth response of CTX-M-1-producing E. coli exposed to cefotaxime and to investigate how blaCTX-M-1 expression at mRNA and protein levels is influenced by cefotaxime concentration, growth phase and gene location (chromosome versus plasmid). METHODS: Two isogenic E. coli strains, MG1655/CTX-M-1 and MG1655/IncI1/CTX-M-1, containing blaCTX-M-1 on the chromosome and on a wild-type IncI1 plasmid, respectively, were constructed and the MIC of cefotaxime was determined. Growth of the two strains was studied in the presence of increasing concentrations of cefotaxime ranging from 0 to 512 mg/L. The levels of mRNA and protein in different growth phases and at different cefotaxime concentrations were studied by qPCR and selected-reaction-monitoring MS, respectively. RESULTS: The MICs of cefotaxime were 168 and 252 mg/L for MG1655/CTX-M-1 and MG1655/IncI1/CTX-M-1, respectively. Both strains displayed a prolonged lag phase when exposed to cefotaxime. The mRNA of blaCTX-M-1 and CTX-M-1 protein levels increased in the presence of high cefotaxime concentrations and varied with growth phase. Higher mRNA expression levels were detected for MG1655/CTX-M-1 compared with MG1655/IncI1/CTX-M-1, but a higher protein level was found for MG1655/IncI1/CTX-M-1 compared with MG1655/CTX-M-1, the latter corresponding well with the higher MIC for this strain. CONCLUSIONS: blaCTX-M-1 mRNA expression and CTX-M-1 protein levels were dependent on cefotaxime concentration, growth phase and gene location. These results provide insight into the expression of cephalosporin resistance in CTX-M-1-producing E. coli, improving our understanding of the relationship between antimicrobial therapy and the expression of resistance mechanisms.

Identification of potential drug targets in Salmonella enterica sv. Typhimurium using metabolic modelling and experimental validation.

Salmonella enterica sv. Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of S. Typhimurium and associated databases, a genome-scale metabolic model was constructed. Output was based on an experimental determination of the biomass of Salmonella when growing in glucose minimal medium. Linear programming was used to simulate variations in the energy demand while growing in glucose minimal medium. By grouping reactions with similar flux responses, a subnetwork of 34 reactions responding to this variation was identified (the catabolic core). This network was used to identify sets of one and two reactions that when removed from the genome-scale model interfered with energy and biomass generation. Eleven such sets were found to be essential for the production of biomass precursors. Experimental investigation of seven of these showed that knockouts of the associated genes resulted in attenuated growth for four pairs of reactions, whilst three single reactions were shown to be essential for growth.

Trapping and proteomic identification of cellular substrates of the ClpP protease in Staphylococcus aureus.

In the important human pathogen Staphylococcus aureus the cytoplasmic ClpP protease is essential for mounting cellular stress responses and for virulence. To directly identify substrates of the ClpP protease, we expressed in vivo a proteolytic inactive form of ClpP (ClpP(trap)) that will retain but not degrade substrates translocated into its proteolytic chamber. Substrates captured inside the proteolytic barrel were co-purified along with the His-tagged ClpP complex and identified by mass spectrometry. In total, approximately 70 proteins were trapped in both of the two S. aureus strains NCTC8325-4 and Newman. About one-third of the trapped proteins are previously shown to be unstable or to be substrates of ClpP in other bacteria, supporting the validity of the ClpP-TRAP. This group of proteins encompassed the transcriptional regulators CtsR and Spx, the ClpC adaptor proteins McsB and MecA, and the cell division protein FtsZ. Newly identified ClpP substrates include the global transcriptional regulators PerR and HrcA, proteins involved in DNA damage repair (RecA, UvrA, UvrB), and proteins essential for protein synthesis (RpoB and Tuf). Our study hence underscores the central role of Clp-proteolysis in a number of pathways that contribute to the success of S. aureus as a human pathogen.

Complete Genome Sequences of Extraintestinal Pathogenic Escherichia coli Clinical Isolates from Danish Ulcerative Colitis Patients.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a potential factor in ulcerative colitis etiology. We report here the complete genome and plasmid sequences of three Escherichia coli isolates, C 237-04 (p7), C 236-04A (p10A), and C 691-04A (p19A), obtained from fecal samples from ulcerative colitis patients in Copenhagen, Denmark.

Complete Genome Sequence of a Multidrug-Resistant Klebsiella pneumoniae Environmental Isolate from Zanzibar, Tanzania, Harboring Novel Insertion Elements and Two blaCTX-M-15 Genes.

Here, we report the annotated whole-genome sequence of Klebsiella pneumoniae strain KP_3b, isolated in Zanzibar, Tanzania, from plastic litter. The strain is extended-spectrum ß-lactamase (ESBL) producing and multidrug resistant, encoding 17 resistance genes, most of which are located on a 230,544-bp plasmid. The isolate contains two copies of the blaCTX-M-15 gene and novel insertion elements.

Efficacy of piperacillin-tazobactam and cefotaxime against Escherichia coli hyperproducing TEM-1 in a mouse peritonitis infection model.

OBJECTIVES: Piperacillin-tazobactam (TZP) is a frequently prescribed antibiotic in hospital settings. Reports suggest in vivo efficacy of TZP, despite in vitro resistance of isolates susceptible to cephalosporins. Escherichia coli (E. coli) isolates hyperproducing TEM-1 ß-lactamase possess this phenotype. This study investigated the influence of tazobactam (TAZ) concentration on piperacillin (PIP) inhibition of such isolates and compared the in vivo efficacy of TZP with cefotaxime (CTX) in an infection model. METHODS: The PIP MICs for E. coli isolates, either hyperproducing TEM-1 because of promoter substitutions (n = 4) or because of gene amplification (n = 2) or producing an inhibitor-resistant TEM-35 (IRT) (n = 1), were determined using increasing concentrations of TAZ in a checkerboard setup. Furthermore, the efficacy of TZP and CTX against the isolates was investigated in a mouse peritonitis model using antibiotic exposures mimicking human conditions. Isolates producing either OXA-48 or CTX-M-15 ß-lactamases were included as controls. RESULTS: Using TAZ concentrations ≤ 64 mg/L, one isolate hyperproducing TEM-1 had a PIP MIC of 8 at TAZ 16 mg/L and two additional isolates at TAZ 64 mg/L. In the mouse peritonitis infection model, reduction of bacterial load in the peritoneum was larger for TZP than CTX only for the CTX-M-15-producing isolate. Larger reductions in bacterial load were observed after CTX treatment than TZP treatment for seven of the eight remaining test isolates. CONCLUSIONS: Piperacillin-tazobactam treatment of E. coli isolates hyperproducing TEM-1 was less effective than CTX treatment and may, for some isolates, be comparable with TZP treatment of isolates producing established resistance markers as IRT or OXA-48.

Microplastics in the human digestive environment: A focus on the potential and challenges facing in vitro gut model development.

Plastic pollution is a major issue worldwide, generating massive amounts of smaller plastic particles, including microplastics (MPs). Their ubiquitous nature in the environment but also in foodstuff and consumer packaged goods has revealed potential threats to humans who can be contaminated mainly through air, food and water consumption. In this review, the current literature on human exposure to MPs is summarized with a focus on the gastrointestinal tract as portal of entry. Then, we discuss the vector effect of MPs, in their pristine versus weathered forms, with well-known contaminants as heavy metals and chemicals, or more emerging ones as antibiotics or microbial pathogens, like Pseudomonas spp., Vibrio spp., Campylobacter spp. and Escherichia coli. Comprehensive knowledge on MP fate in the gastrointestinal tract and their potential impact on gut homeostasis disruption, including gut microbiota, mucus and epithelial barrier, is reported in vitro and in vivo in mammals. Special emphasis is given on the crucial need of developing robust in vitro gut models to adequately simulate human digestive physiology and absorption processes. Finally, this review points out future research directions on MPs in human intestinal health.

Isolation and characterization of human pathogenic multidrug resistant bacteria associated with plastic litter collected in Zanzibar.

Plastic pollution is a growing problem, not at least in areas where poor waste management results in direct pollution of coastal zones, such as South Asia and regions in Africa. In addition to the effect on ecosystems and their related services, plastic pollution may also affect human health indirectly as vectors for infectious disease. As plastic offers a suitable surface for the attachment of biofilm forming bacteria, it may contribute to disease outbreaks and antimicrobial resistance. To investigate the role of plastic litter as potential vectors for pathogenic bacteria, we collected plastic litter from four rural sites in Zanzibar, and isolated adhered bacteria. Isolates were short-read sequenced for further molecular analysis. This revealed that collected plastic litter was associated with diverse bacterial species, including human pathogens Citrobacter freundii, Klebsiella pneumoniae and Vibrio cholerae. Furthermore, most isolates were found to be multidrug resistant. Our findings confirm that plastic litter, serve as novel reservoir for human multidrug resistant pathogenic bacteria that combined with poor sanitation and waste handling, may lead to transmission of infectious diseases and antimicrobial resistance. These findings add a new level to the environmental challenges with plastic pollution; the potential health risk associated with exposure to plastic litter.

Growth phase-dependent regulation of the global virulence regulator Rot in clinical isolates of Staphylococcus aureus.

Current models for global virulence regulation in Staphylococcus aureus are mainly based on studies performed with only a limited number of laboratory strains derived from NCTC8325. In these strains the small regulatory RNA, RNAIII, has a central role in virulence gene regulation. Recently, RNAIII was suggested to control transcription of target genes partly by inhibiting translation of the transcriptional regulator Rot. The present study was undertaken to examine if the model for RNAIII/Rot-dependent virulence regulation is conserved among clinical strains. To this end, we used Rot antibodies to directly assess the amount of Rot protein in 4 well-characterized S. aureus laboratory strains (8325-4, COL, Newman, and UAMS-1) and in 9 strains of clinical origin (encompassing USA300 and Mu50). Additionally, the cellular amount of RNAIII and rot mRNA was determined in all strains. The experiments revealed considerable variation in the Rot and RNAIII levels between strains. However, in the majority of strains the cellular amount of Rot was inversely correlated to the RNAIII level. As we demonstrate that Rot is a stable protein and that the level of rot transcript appeared similar in all strains, our data support that the model for RNAIII-mediated inhibition of rot mRNA translation is conserved among clinical strains. Assessment of Rot-dependent regulation of target genes revealed that Rot is a positive regulator of spa (protein A) transcription in all strains examined. In contrast, Rot repression of sspA (serine protease) and hlb (beta-hemolysin) transcription was not conserved between strains. From this study, we conclude that while the paradigm for understanding RNAIII-dependent regulation of Rot is well-conserved, regulation of single genes is subject to considerable strain variation. We propose that variation in global regulatory networks contribute considerably to the phenotypic variation observed between S. aureus isolates.

Complete genome sequence of the myxobacterium Sorangium cellulosum.

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.

Polyamine depletion has global effects on stress and virulence gene expression and affects HilA translation in Salmonella enterica serovar typhimurium.

Polyamines are small cationic amines required for modulating multiple cell process, including cell growth and DNA and RNA stability. In Salmonella polyamines are primarily synthesized from L-arginine or L-ornithine. Based on a previous study, which demonstrated that polyamines affect the expression of virulence gene in S. Typhimurium, we investigated the role of polyamines in the global gene and protein expression in S. Typhimurium. The depletion of polyamine biosynthesis led to down-regulation of genes encoding structural components of the Type Three Secretion system 1 (TTSS1) and its secreted effectors. Interestingly, Expression of HilA, which is the master regulator of Salmonella Pathogenicity Island 1 (SPI1), was only reduced at the post-transcriptional in the polyamine mutant. Enzymes related to biosynthesis and/or transport of several amino acids were up-regulated, just as the Mg2+-transport systems were three to six-fold up-regulated at both the transcriptional and protein levels. Furthermore, in the polyamine depletion mutant, proteins related to stress response (IbpA, Dps, SodB), were 2-5 fold up-regulated. Together our data provide strong evidence that polyamine depletion affects expression of proteins linked with virulence and stress response of S. Typhimurium. Furthermore, polyamines positively affected translation of HilA, the major regulator of SPI1.

The SPI-19 encoded type-six secretion-systems (T6SS) of Salmonella enterica serovars Gallinarum and Dublin play different roles during infection.

Salmonella Pathogenicity Islands 19 (SPI19) encodes a type VI secretion system (T6SS). SPI19 is only present in few serovars of S. enterica, including the host-adapted serovar S. Dublin and the host-specific serovar S. Gallinarum. The role of the SPI19 encoded T6SS in virulence in these serovar is not fully understood. Here we show that during infection of mice, a SPI19/T6SS deleted strain of S. Dublin 2229 was less virulent than the wild type strain after oral challenge, but not after IP challenge. The mutant strain also competed significantly poorer than the wild type strain when co-cultured with strains of E. coli, suggesting that this T6SS plays a role in pathogenicity by killing competing bacteria in the intestine. No significant difference was found between wild type S. Gallinarum G9 and its ΔSPI19/T6SS mutant in infection, whether chicken were challenged orally or by the IP route, and the S. Gallinarum G9 ΔSPI19/T6SS strain competed equally well as the wild type strain against strains of E. coli. However, contrary to what was observed with S. Dublin, the wild type G9 strains was significantly more cytotoxic to monocyte derived primary macrophages from hens than the mutant, suggesting that SPI19/T6SS in S. Gallinarum mediates killing of eukaryotic cells. The lack of significant importance of SPI19/T6SS after oral and systemic challenge of chicken was confirmed by knocking out SPI19 in a second strain, J91. Together the results suggest that the T6SS encoded from SPI19 have different roles in the two serovars and that it is a virulence-factor after oral challenge of mice in S. Dublin, while we cannot confirm previous results that SPI19/T6SS influence virulence significantly in S. Gallinarum.