Isolation of Spectinomycin Resistance Mutations in the 16S rRNA of Salmonella enterica serovar Typhimurium and Expression in Escherichia coli and Salmonella

Two single-base mutations in 16S rRNA conferring high-level resistance to spectinomycin were isolated on a plasmid-borne copy of the rrnD operon from Salmonella enterica serovar Typhimurium. Neither of the mutations (C1066U and C1192U) had appreciable effects on cell growth, but each had differential effects on resistance to spectinomycin and fusidic acid. Both mutations also conferred resistance to spectinomycin in Escherichia coli strains containing deletions of all seven chromosomal rrn operons and expressing plasmid-encoded Salmonella rRNA exclusively. In contrast, when expressed in E. coli strains containing intact chromosomal rrn operons, the strains were sensitive to spectinomycin. However, chromosomal mutations arose that allowed expression of the rRNA-dependent spectinomycin resistance phenotype. It is proposed that in heterogeneous rRNA populations, the native E. coli rRNA out-competes the heterologous Salmonella rRNA for binding to ribosomal proteins, translation factors, or ribosome assembly, thus limiting entry of the antibiotic-resistant 30S subunits into the functioning ribosome pool. Received: 28 September 2001 / Accepted: 26 March 2002     

OmpR May Regulate the Putative YehU/YehT Two-Component System in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhi Under Hypotonic Growth Condition

Decreased expression (twofold) of a putative yehUTS operon of which yehUT encodes a putative YehU/YehT two-component system in the ompR mutant from Salmonella enterica serovar Typhi (S. Typhi) GIFU10007 under hypotonic growth condition was observed by qRT-PCR. Purified recombinant protein OmpR_His6 of GIFU10007 was shown to bind the upstream region of the yehU gene by the gel-shift assay. In addition, the yehT deletion mutant (ΔyehT) displayed differential expression (twofold or higher) of 26 genes under the condition by the DNA microarray analysis. Altogether, OmpR might regulate the YehUT system in S. Typhi under hypotonic growth condition.     

The Vi Capsular Polysaccharide Enables Salmonella enterica Serovar Typhi to Evade Microbe-Guided Neutrophil Chemotaxis

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.     

Comparative Proteomic Analysis of the PhoP Regulon in Salmonella enterica Serovar Typhi Versus Typhimurium

Background

S. Typhi, a human-restricted Salmonella enterica serovar, causes a systemic intracellular infection in humans (typhoid fever). In comparison, S. Typhimurium causes gastroenteritis in humans, but causes a systemic typhoidal illness in mice. The PhoP regulon is a well studied two component (PhoP/Q) coordinately regulated network of genes whose expression is required for intracellular survival of S. enterica.

Methodology/Principal Findings

Using high performance liquid chromatography mass spectrometry (HPLC-MS/MS), we examined the protein expression profiles of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro and compared these results to profiles of phoP⁻/Q⁻ mutants derived from S. Typhimurium LT2 and S. Typhi Ty2. Our analysis identified 53 proteins in S. Typhimurium LT2 and 56 proteins in S. Typhi that were regulated in a PhoP-dependent manner. As expected, many proteins identified in S. Typhi demonstrated concordant differential expression with a homologous protein in S. Typhimurium. However, three proteins (HlyE, STY1499, and CdtB) had no homolog in S. Typhimurium. HlyE is a pore-forming toxin. STY1499 encodes a stably expressed protein of unknown function transcribed in the same operon as HlyE. CdtB is a cytolethal distending toxin associated with DNA damage, cell cycle arrest, and cellular distension. Gene expression studies confirmed up-regulation of mRNA of HlyE, STY1499, and CdtB in S. Typhi in PhoP-inducing conditions.

Conclusions/Significance

This study is the first protein expression study of the PhoP virulence associated regulon using strains of Salmonella mutant in PhoP, has identified three Typhi-unique proteins (CdtB, HlyE and STY1499) that are not present in the genome of the wide host-range Typhimurium, and includes the first protein expression profiling of a live attenuated bacterial vaccine studied in humans (Ty800).     

Salmonella enterica Serovar Typhi Conceals the Invasion-Associated Type Three Secretion System from the Innate Immune System by Gene Regulation

Delivery of microbial products into the mammalian cell cytosol by bacterial secretion systems is a strong stimulus for triggering pro-inflammatory host responses. Here we show that Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, tightly regulates expression of the invasion-associated type III secretion system (T3SS-1) and thus fails to activate these innate immune signaling pathways. The S. Typhi regulatory protein TviA rapidly repressed T3SS-1 expression, thereby preventing RAC1-dependent, RIP2-dependent activation of NF-κB in epithelial cells. Heterologous expression of TviA in S. enterica serovar Typhimurium (S. Typhimurium) suppressed T3SS-1-dependent inflammatory responses generated early after infection in animal models of gastroenteritis. These results suggest that S. Typhi reduces intestinal inflammation by limiting the induction of pathogen-induced processes through regulation of virulence gene expression.     

Extracellular DNA Inhibits Salmonella enterica Serovar Typhimurium and S. enterica Serovar Typhi Biofilm Development on Abiotic Surfaces

Extracellular DNA (eDNA) was identified and characterized in a 2-day-old biofilms developed by Salmonella enterica ser. Typhimurium SR-11 and S. enterica ser. Typhi ST6 using confocal laser scanning microscopy (CLSM) and enzymatic extraction methods. Results of microtitre plate assay and CLSM analysis showed both Salmonella strains formed significantly more biofilms in the presence of DNase I; Furthermore, a remarkable decrease of biofilm formation was observed when eDNA was added in the inoculation. However, for the pre-established biofilms on polystyrene and glass, no significant difference was observed between the DNase I treated biofilm and the corresponding non-treated controls. In conclusion, these results demonstrate that eDNA is a novel matrix component of Salmonella biofilms. This is the first evidence for the presence of eDNA and its inhibitive and destabilizing effect during biofilm development of S. enterica ser. Typhimurium and S. enterica ser. Typhi on abiotic surfaces.     

Kinetic model‐based prediction of the persistence of Salmonella enterica serovar Typhimurium under tropical agricultural field conditions

Aim: Present a kinetic model‐based approach for using isothermal data to predict the survival of manure‐borne enteric bacteria under dynamic conditions in an agricultural environment. Methods and Results: A model to predict the survival of Salmonella enterica serovar Typhimurium under dynamic temperature conditions in soil in the field was developed. The working hypothesis was that the inactivation phenomena associated with the survival kinetics of an organism in an agricultural matrix under dynamic temperature conditions is for a large part due to the cumulative effect of inactivation at various temperatures within the continuum registered in the matrix in the field. The modelling approach followed included (i) the recording of the temperature profile that the organism experiences in the field matrix, (ii) modelling the survival kinetics under isothermal conditions at a range of temperatures that were registered in the matrix in the field; and (iii) using the isothermal‐based kinetic models to develop models for predicting survival under dynamic conditions. The time needed for 7 log CFU g^?1Salmonella Typhimurium in manure and manure‐amended soil to reach the detection limit of the enumeration method (2 log CFU g^?1) under tropical conditions in the Central Agro‐Ecological Zone of Uganda was predicted to be 61–68 days and corresponded with observed CFU of about 2·2–3·0 log CFU g^?1, respectively. The Bias and Accuracy factor of the prediction was 0·71–0·84 and 1·2–1·4, respectively. Conclusions: Survival of Salm. Typhimurium under dynamic field conditions could be for 71–84% determined by the developed modelling approach, hence substantiating the working hypothesis. Significance and Impact of the Study: Survival kinetic models obtained under isothermal conditions can be used to develop models for predicting the persistence of manure‐borne enteric bacteria under dynamic field conditions in an agricultural environment.     

Host–Pathogen Interaction in Invasive Salmonellosis

Salmonella enterica infections result in diverse clinical manifestations. Typhoid fever, caused by S. enterica serovar Typhi (S. Typhi) and S. Paratyphi A, is a bacteremic illness but whose clinical features differ from other Gram-negative bacteremias. Non-typhoidal Salmonella (NTS) serovars cause self-limiting diarrhea with occasional secondary bacteremia. Primary NTS bacteremia can occur in the immunocompromised host and infants in sub-Saharan Africa. Recent studies on host–pathogen interactions in Salmonellosis using genome sequencing, murine models, and patient studies have provided new insights. The full genome sequences of numerous S. enterica serovars have been determined. The S. Typhi genome, compared to that of S. Typhimurium, harbors many inactivated or disrupted genes. This can partly explain the different immune responses both serovars induce upon entering their host. Similar genome degradation is also observed in the ST313 S. Typhimurium strain implicated in invasive infection in sub-Saharan Africa. Virulence factors, most notably, type III secretion systems, Vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and various factors essential for the intracellular life cycle of S. enterica have been characterized. Genes for these factors are commonly carried on Salmonella Pathogenicity Islands (SPIs). Plasmids also carry putative virulence-associated genes as well as those responsible for antimicrobial resistance. The interaction of Salmonella pathogen-associated molecular patterns (PAMPs) with Toll-like receptors (TLRs) and NOD-like receptors (NLRs) leads to inflammasome formation, activation, and recruitment of neutrophils and macrophages and the production of pro-inflammatory cytokines, most notably interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and interferon-gamma (IFN)-γ. The gut microbiome may be an important modulator of this immune response. S. Typhimurium usually causes a local intestinal immune response, whereas S. Typhi, by preventing neutrophil attraction resulting from activation of TLRs, evades the local response and causes systemic infection. Potential new therapeutic strategies may lead from an increased understanding of infection pathogenesis.     

Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells

Cytolethal‐distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram‐negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella‐containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71‐CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71‐CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71‐CDT also secreted OMVs‐like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin‐loaded OMVs by bystander cells was dependent on dynamin‐1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria.     

伤寒沙门菌基因组DNA芯片的制备与基因表达谱分析应用

伤寒沙门菌是一种具有鞭毛的革兰阴性人类肠道致病菌,也是一种重要的原核生物研究用模式菌．基因组芯片能够系统、全面且高效地观察生物的基因表达及进行基因组结构比较．利用伤寒沙门菌现有的全基因组序列,以Ty2菌株的基因组为基准,选取CT18菌株和z66阳性菌株的特异性蛋白编码基因,设计特异性引物,经PCR有效扩增出4 201个基因,产物纯化后点样于多聚赖氨酸玻片制备伤寒沙门菌基因组DNA芯片,并验证了芯片样点位次与效果．通过对基因表达谱分析的各种条件进行优化,建立相应的表达谱分析方法,并用于比较伤寒沙门菌野生株在高渗、低渗条件下的基因表达差异,结果与以前的报道基本一致．结果表明,成功建立了伤寒沙门菌基因组DNA芯片及表达谱分析方法,可为有关伤寒沙门菌基因表达调控及致病性机理、进化和基因多样性等方面的深入研究提供有效的技术支持．     

Bloodstream Infection among Children Presenting to a General Hospital Outpatient Clinic in Urban Nepal

Background

There are limited data on the etiology and characteristics of bloodstream infections in children presenting in hospital outpatient settings in South Asia. Previous studies in Nepal have highlighted the importance of murine typhus as a cause of febrile illness in adults and enteric fever as a leading bacterial cause of fever among children admitted to hospital.

Methods

We prospectively studied a total of 1084 febrile children aged between 2 months and 14 years presenting to a general hospital outpatient department in Kathmandu Valley, Nepal, over two study periods (summer and winter). Blood from all patients was tested by conventional culture and by real-time PCR for Rickettsia typhi.

Results

Putative etiological agents for fever were identified in 164 (15%) patients. Salmonella enterica serovar Typhi (S. Typhi) was identified in 107 (10%), S. enterica serovar Paratyphi A (S. Paratyphi) in 30 (3%), Streptococcus pneumoniae in 6 (0.6%), S. enterica serovar Typhimurium in 2 (0.2%), Haemophilus influenzae type b in 1 (0.1%), and Escherichia coli in 1 (0.1%) patient. S. Typhi was the most common organism isolated from blood during both summer and winter. Twenty-two (2%) patients were PCR positive for R. typhi. No significant demographic, clinical and laboratory features distinguished culture positive enteric fever and murine typhus.

Conclusions

Salmonella infections are the leading cause of bloodstream infection among pediatric outpatients with fever in Kathmandu Valley. Extension of immunization programs against invasive bacterial disease to include the agents of enteric fever and pneumococcus could improve the health of children in Nepal.     

<Emphasis Type="Italic">S</Emphasis>. Typhimurium <Emphasis Type="Italic">sseJ</Emphasis> gene decreases the <Emphasis Type="Italic">S</Emphasis>. Typhi cytotoxicity toward cultured epithelial cells

Background  

Salmonella enterica serovar Typhi and Typhimurium are closely related serovars as indicated by >96% DNA sequence identity between shared genes. Nevertheless, S. Typhi is a strictly human-specific pathogen causing a systemic disease, typhoid fever. In contrast, S. Typhimurium is a broad host range pathogen causing only a self-limited gastroenteritis in immunocompetent humans. We hypothesize that these differences have arisen because some genes are unique to each serovar either gained by horizontal gene transfer or by the loss of gene activity due to mutation, such as pseudogenes. S. Typhi has 5% of genes as pseudogenes, much more than S. Typhimurium which contains 1%. As a consequence, S. Typhi lacks several protein effectors implicated in invasion, proliferation and/or translocation by the type III secretion system that are fully functional proteins in S. Typhimurium. SseJ, one of these effectors, corresponds to an acyltransferase/lipase that participates in SCV biogenesis in human epithelial cell lines and is needed for full virulence of S. Typhimurium. In S. Typhi, sseJ is a pseudogene. Therefore, we suggest that sseJ inactivation in S. Typhi has an important role in the development of the systemic infection.