Complete Genome Sequence of a Multidrug-Resistant <i>Klebsiella pneumoniae</i> Environmental Isolate from Zanzibar, Tanzania, Harboring Novel Insertion Elements and Two <i>bla</i>CTX-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 β-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 bla(CTX-M-15) gene and novel insertion elements

The putative thiosulfate sulfurtransferases PspE and GlpE contribute to virulence of <em>Salmonella</em> Typhimurium in the mouse model of systemic disease.

The phage-shock protein PspE and GlpE of the glycerol 3-phosphate regulon of Salmonella enterica serovar Typhimurium are predicted to belong to the class of thiosulfate sulfurtransferases, enzymes that traffic sulfur between molecules. In the present study we demonstrated that the two genes contribute to S. Typhimurium virulence, as a glpE and pspE double deletion strain showed significantly decreased virulence in a mouse model of systemic infection. However, challenge of cultured epithelial cells and macrophages did not reveal any virulence-associated phenotypes. We hypothesized that their contribution to virulence could be in sulfur metabolism or by contributing to resistance to nitric oxide, oxidative stress, or cyanide detoxification. In vitro studies demonstrated that glpE but not pspE was important for resistance to H(2)O(2). Since the double mutant, which was the one affected in virulence, was not affected in this assay, we concluded that resistance to oxidative stress and the virulence phenotype was most likely not linked. The two genes did not contribute to nitric oxid stress, to synthesis of essential sulfur containing amino acids, nor to detoxification of cyanide. Currently, the precise mechanism by which they contribute to virulence remains elusive

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 C(1) units in vivo (CI = 0.03 ± 0.03). In contrast, GlyA was the only important enzyme for the formation of C(1) units in vitro. The results with the ΔgcvT mutant further revealed that formation of serine by SerA and further conversion of serine into C(1) units and glycine by GlyA were not sufficient to ensure C(1) 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

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.Priscila R. Guerra was supported by a scholarship from CAPES – Brazilian Federal Agency for Support and Evaluation of Graduate Education within the Ministry of Education of Brazil. This study received financial support from Spanish Ministry of Economy, Industry and Competitiveness (AGL2013–45431-R and AGL2016–78708-R), and from the Danish Council for Independent Research (Technology and Production), grant number DFF – 4184-00050. The sponsors had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Removal of the phage-shock protein PspB causes reduction of virulence in Salmonella enterica serovar Typhimurium independently of NRAMP1

The phage-shock protein (Psp) system is believed to manage membrane stress in all Enterobacteriaceae and has recently emerged as being important for virulence in several pathogenic species of this phylum. The core of the Psp system consists of the pspA-D operon and the distantly located pspG gene. In Salmonella enterica serovar Typhimurium (S. Typhimurium), it has recently been reported that PspA is essential for systemic infection of mice, but only in NRAMP1(+) mice, signifying that attenuation is related to coping with divalent cation starvation in the intracellular environment. In the present study, we investigated the contribution of individual psp genes to virulence of S. Typhimurium. Interestingly, deletion of the whole pspA-D set of genes caused attenuation in both NRAMP1(+) and NRAMP1(-) mice, indicating that one or more of the psp genes contribute to virulence independently of NRAMP1 expression in the host. Investigations of single gene mutants showed that knock out of pspB reduced virulence in both types of mice, while deletion of pspA only caused attenuation in NRAMP1(+) mice, and deletion of pspD had a minor effect in NRAMP1(-) mice, while deletions of either pspC or pspG did not affect virulence. Experiments addressed at elucidating the role of PspB in virulence revealed that PspB is dispensable for uptake to and intracellular replication in cultured macrophages and resistance to complement-induced killing. Furthermore, the Psp system of S. Typhimurium was dispensable during pIV-induced secretin stress. In conclusion, our results demonstrate that removal of PspB reduces virulence in S. Typhimurium independently of host NRAMP1 expression, demonstrating that PspB has roles in intra-host survival distinct from the reported contributions of PspA

Identification of Metabolic Pathways Essential for Fitness of <i>Salmonella</i> Typhimurium <i>In Vivo</i>

Bacterial infections remain a threat to human and animal health worldwide, and there is an urgent need to find novel targets for intervention. In the current study we used a computer model of the metabolic network of Salmonella enterica serovar Typhimurium and identified pairs of reactions (cut sets) predicted to be required for growth in vivo. We termed such cut sets synthetic auxotrophic pairs. We tested whether these would reveal possible combined targets for new antibiotics by analyzing the performance of selected single and double mutants in systemic mouse infections. One hundred and two cut sets were identified. Sixty-three of these included only pathways encoded by fully annotated genes, and from this sub-set we selected five cut sets involved in amino acid or polyamine biosynthesis. One cut set (asnA/asnB) demonstrated redundancy in vitro and in vivo and showed that asparagine is essential for S. Typhimurium during infection. trpB/trpA as well as single mutants were attenuated for growth in vitro, while only the double mutant was a cut set in vivo, underlining previous observations that tryptophan is essential for successful outcome of infection. speB/speF,speC was not affected in vitro but was attenuated during infection showing that polyamines are essential for virulence apparently in a growth independent manner. The serA/glyA cut-set was found to be growth attenuated as predicted by the model. However, not only the double mutant, but also the glyA mutant, were found to be attenuated for virulence. This adds glycine production or conversion of glycine to THF to the list of essential reactions during infection. One pair (thrC/kbl) showed true redundancy in vitro but not in vivo demonstrating that threonine is available to the bacterium during infection. These data add to the existing knowledge of available nutrients in the intra-host environment, and have identified possible new targets for antibiotics