Salmonella enterica serovar Typhimurium is a pathogen harbored by livestock and shed in their feces, which serves as an acquisition source for adult house flies. This study used a green fluorescent protein (GFP) expressing strain of Salmonella Typhimurium to assess its acquisition by and survival within house flies, and transmission from and between flies in the presence or absence of cantaloupe. Female house flies were exposed to manure inoculated with either sterile phosphate-buffered saline or GFP-Salmonella Typhimurium for 12 h, then used in four experiments each performed over 24 h. Experiment 1 assessed the survival of GFP-Salmonella Typhimurium within inoculated flies. Experiment 2 determined transmission of GFP-Salmonella Typhimurium from inoculated flies to cantaloupe. Experiment 3 assessed fly acquisition of GFP-Salmonella Typhimurium from inoculated cantaloupe. Experiment 4 evaluated transmission of GFP-Salmonella Typhimurium between inoculated flies and uninoculated flies in the presence and absence of cantaloupe. GFP-Salmonella Typhimurium survived in inoculated flies but bacterial abundance decreased between 0 and 6 h without cantaloupe present and between 0 and 6 h and 6 and 24 h with cantaloupe present. Uninoculated flies acquired GFP-Salmonella Typhimurium from inoculated cantaloupe and bacterial abundance increased in cantaloupe and flies from 6 to 24 h. More uninoculated flies exposed to inoculated flies acquired GFP-Salmonella Typhimurium when cantaloupe was present than when absent. We infer that the presence of a shared food source facilitated the transfer of GFP-Salmonella Typhimurium from inoculated to uninoculated flies. Our study demonstrated that house flies acquired, harbored, and excreted viable GFP-Salmonella Typhimurium and transferred bacteria to food and each other. Understanding the dynamics of bacterial acquisition and transmission of bacteria between flies and food helps in assessing the risk flies pose to food safety and human health.

• Klíčová slova
• Musca domestica, Salmonella enterica, food safety, house flies, pathogen transmission, vector potential,
• MeSH
• Cucumis melo mikrobiologie   MeSH
• kontaminace potravin analýza   MeSH
• moucha domácí mikrobiologie   MeSH
• potravinářská mikrobiologie metody   MeSH
• Salmonella typhimurium patogenita   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• zelené fluorescenční proteiny MeSH

Peptidyl-prolyl cis-trans isomerases (PPIase) exhibit chaperone activity and assist in protein folding by increasing the rate of cis-trans transition on proline-peptide bonds. The current study aimed to identify and characterize three genes, ppiA, ppiB, and ppiC, which encode proteins of the PPIase family in the bacterium Salmonella enterica serovar Typhimurium. Salmonella Typhimurium is a facultative intracellular zoonotic pathogen that causes food- and water-borne gastroenteritis in humans (leading to bacteremia in immune-compromised subjects). Recombinant clones for the three genes were constructed and sequenced and the sequences submitted to NCBI GenBank. Three-dimensional structures for the corresponding proteins were predicted by comparative modeling. A maximum-likelihood phylogenetic gene tree constructed for the three genes showed a low evolutionary mean diversity, indicating strong evolutionary conservation. Further, single-gene deletion mutant strains, generated for the respective genes, were observed to be more susceptible to the stationary phase of growth and heat stress conditions and showed reduced survival within macrophage cells line. The present study thus indicates that ppiA, ppiB, and ppiC genes are conserved among Salmonella genome, are critical for the growth of Salmonella Typhimurium in the examined stress conditions, and may play a role in its responses and virulence.

• Klíčová slova
• Peptidyl-prolyl cis-trans isomerases, Salmonella Typhimurium, ppiA, ppiB, ppiC and stress responses,
• MeSH
• bakteriální proteiny chemie   genetika   MeSH
• fylogeneze * MeSH
• fyziologický stres * MeSH
• genom bakteriální MeSH
• kur domácí MeSH
• peptidylprolylisomerasa chemie   genetika   MeSH
• Salmonella typhimurium enzymologie   genetika   patogenita   MeSH
• salmonelová infekce u zvířat mikrobiologie   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• bakteriální proteiny MeSH
• peptidylprolylisomerasa MeSH

Conjugative plasmids from the IncHI1 incompatibility group play an important role in transferring antibiotic resistance in Salmonella Typhimurium. However, knowledge of their genome structure or gene expression is limited. In this study, we determined the complete nucleotide sequences of four IncHI1 plasmids transferring resistance to antibiotics by two different next generation sequencing protocols and protein expression by mass spectrometry. Sequence data including additional 11 IncHI1 plasmids from GenBank were used for the definition of the IncHI1 plasmid core-genome and pan-genome. The core-genome consisted of approximately 123 kbp and 122 genes while the total pan-genome represented approximately 600 kbp. When the core-genome sequences were used for multiple alignments, the 15 tested IncHI1 plasmids were separated into two main lineages. GC content in core-genome genes was around 46% and 50% in accessory genome genes. A multidrug resistance region present in all 4 sequenced plasmids extended over 20 kbp and, except for tet(B), the genes responsible for antibiotic resistance were those with the highest GC content. IncHI1 plasmids therefore represent replicons that evolved in low GC content bacteria. From their original host, they spread to Salmonella and during this spread these plasmids acquired multiple accessory genes including those coding for antibiotic resistance. Antibiotic-resistance genes belonged to genes with the highest level of expression and were constitutively expressed even in the absence of antibiotics. This is the likely mechanism that facilitates host cell survival when antibiotics suddenly emerge in the environment.

• Klíčová slova
• IncHI1 plasmids, comparative genomic, proteomics,
• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence genetika   MeSH
• genom bakteriální genetika   MeSH
• interakce hostitele a patogenu genetika   MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• plazmidy genetika   MeSH
• regulace genové exprese u bakterií účinky léků   MeSH
• Salmonella typhimurium účinky léků   genetika   patogenita   MeSH
• salmonelóza genetika   mikrobiologie   MeSH
• sekvenční analýza DNA MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky MeSH

In this study we determined the complete nucleotide sequence of multidrug-resistance plasmid p9134, and its variants p9134dT and p9134dAT which spontaneously lost either tetracycline or both tetracycline and ampicillin resistance, respectively. The plasmids were 133,802 bp, 109,512 bp and 127,291 bp in size, respectively, and their basic backbone was similar to that of IncI plasmids. Genes coding for ampicillin (blaTEM), chloramphenicol (catA1), streptomycin (strA, strB), tetracycline (tetA(A)) and gentamicin (aac(3)-IV) resistance were confirmed in wild-type p9134. Moreover, a gene for hygromycine resistance (hph) and a putative gene for apramycin resistance were newly determined. In p9134dAT, a continuous sequence coding for ampicillin and tetracycline resistances was lost. Genetic rearrangements in p9134dT were more complex and 2 recombination events must have occurred. During the first one, the tetracycline resistance locus was replaced with rck, srgB, srgA, orf7 and pefI originating from Salmonella virulence plasmid pSLT. During the second one, ydjA, pifA and repC genes from p9134 were replaced with repA2, PSLT025 and PSLT026 genes from pSLT. Our findings indicate that recombination event between unrelated plasmids might be quite common and may lead to the generation and selection of plasmids both transferring antibiotic resistance and increasing virulence of their host.

• Klíčová slova
• Antibiotic resistance, Conjugation, Plasmid sequence, Recombination, Salmonella,
• MeSH
• mnohočetná bakteriální léková rezistence genetika   MeSH
• plazmidy genetika   MeSH
• rekombinace genetická MeSH
• rezistence na tetracyklin genetika   MeSH
• Salmonella typhimurium účinky léků   genetika   patogenita   MeSH
• sekvenční analýza DNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Salmonella enterica serovar Typhimurium requires the type III secretion system encoded by Salmonella pathogenicity island 1 (SPI1) and controlled by the master regulator, HilA, to penetrate the intestinal epithelium. Numerous regulators affect virulence through influence on this system, including the proteolytic component ClpP, the stationary phase regulator RpoS and the carbon-storage regulator CsrA. However, the mechanism behind the ClpP regulation is not fully understood. To elucidate this we examined differentially expressed genes in a ΔclpP mutant compared with WT using global transcriptomic analysis. SPI1 and SPI4 virulence genes were significantly downregulated in the ΔclpP mutant, whereas several RpoS-dependent genes and the fliC gene encoding flagellin were upregulated. While the ΔclpP mutant was attenuated in cell invasion, this attenuation was not present in a ΔclpP/rpoS : : amp double mutant, suggesting the repression of invasion was directed through RpoS. The expression of the csrA virulence regulator was increased in the ΔclpP mutant and decreased in the rpoS : : amp and ΔclpP/rpoS : : amp mutants, indicating that ClpP affects the csrA expression level as well. Thus, this study suggests that ClpP affects SPI1 expression and thereby virulence indirectly through its regulation of both RpoS and CsrA.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčné linie MeSH
• delece genu * MeSH
• endopeptidasa Clp genetika   metabolismus   MeSH
• epitelové buňky mikrobiologie   MeSH
• lidé MeSH
• makrofágy mikrobiologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• represorové proteiny genetika   metabolismus   MeSH
• Salmonella typhimurium genetika   patogenita   MeSH
• sigma faktor genetika   metabolismus   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• endopeptidasa Clp MeSH
• proteiny vázající RNA MeSH
• represorové proteiny MeSH
• sigma factor KatF protein, Bacteria MeSH Prohlížeč
• sigma faktor MeSH
• Spi1 protein, Salmonella MeSH Prohlížeč

Natural variation in the presence or the absence of STM0517-0529 genes allowing allantoin utilisation has been described in field isolates of the multidrug resistant Salmonella enterica serovar Typhimurium belonging to the phage type DT104. Interestingly, S. enterica subspecies enterica serovar Typhimurium DT104 is quite frequent in pigs and cattle, but rarely present in egg-laying hens. Taking into account the different mode of allantoin metabolism in birds and mammals, we were interested in whether the absence of STM0517-0529 genes may disable this clone in poultry colonisation. We have therefore constructed the allB (also designated as STM0523) mutants in S. enterica subspecies enterica serovar Typhimurium and S. enterica subspecies enterica serovar Enteritidis, and with these, we infected mice, newly hatched chickens and adult egg-laying hens to show that the defect in allantoin utilisation does not influence S. enterica virulence for mice or adult hens, but slightly decreases virulence of S. enterica for chickens. The decrease in virulence of the allB mutant was relatively minor as it could be observed only after a mixed infection model, consistent with a lower prevalence, but not a total absence of such clones in poultry flocks.

• MeSH
• alantoin metabolismus   MeSH
• drůbež MeSH
• kyselina močová metabolismus   MeSH
• mnohočetná bakteriální léková rezistence MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nemoci drůbeže mikrobiologie   patologie   MeSH
• Salmonella enteritidis genetika   metabolismus   patogenita   MeSH
• Salmonella typhimurium genetika   metabolismus   patogenita   MeSH
• salmonelová infekce u zvířat mikrobiologie   patologie   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alantoin MeSH
• kyselina močová MeSH

The role of MicA (repressing small regulatory non-coding RNAs of two Salmonella porins) was determined in virulence of Salmonella enterica serovar Typhimurium. Transcriptional analysis revealed that the expression of the micA gene is driven by a single σ(E)-dependent promoter, micAp. Its activity increased towards stationary phase; in exponential phase, the activity was induced by several stresses by a DegS-dependent mechanism. Although phenotypic analysis revealed no significant differences between wild-type and the micA mutant strains, in vivo studies showed that this mutant is more virulent in the mouse model.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• bakteriální RNA genetika   metabolismus   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nekódující RNA genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• Salmonella typhimurium genetika   metabolismus   patogenita   MeSH
• salmonelóza mikrobiologie   MeSH
• sekvence nukleotidů MeSH
• sigma faktor genetika   metabolismus   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bakteriální RNA MeSH
• nekódující RNA MeSH
• sigma faktor MeSH

OBJECTIVES: In this study, we analysed field isolates of Salmonella enterica serovar Typhimurium for the presence of conjugative plasmids transferring resistances to antibiotics. METHODS: Altogether 23 strains were analysed for the presence of conjugative R-plasmids. In the case of successful conjugation, the R-plasmids were characterized by PCR for antibiotic resistance genes, integrons and replicon typing. Variable regions of integrons were sequenced. RESULTS: Conjugation and transfer of antibiotic resistance was observed in 12 strains. Conjugative plasmids in these strains belonged to the IncI1 and IncHI1 replicons and four of them transferred antibiotic resistance associated with class I integrons. In two cases, resistance to tetracycline and/or ampicillin was not transferred by conjugation to approximately 10% of the transconjugants. Detailed characterization showed that the loss of both resistances was associated with the loss of Tn3 (bla(TEM)) and Tn1721 [tet(A)] from the conjugative plasmids p9046 and p9134. However, when only the tetracycline resistance was lost, the Tn1721 was replaced with a partial sequence of rck, and with complete coding sequences of srgA, srgB, ORF7 and pefI originating from the Salmonella Typhimurium virulence plasmid. CONCLUSIONS: Two plasmids from our collection were capable of recombination with the virulence plasmid of Salmonella Typhimurium and subsequently spread both antibiotic resistance and virulence genes to the recipient.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence * MeSH
• DNA bakterií genetika   MeSH
• faktory virulence genetika   MeSH
• genotyp MeSH
• genová přestavba MeSH
• integrony MeSH
• konjugace genetická MeSH
• lidé MeSH
• plazmidy * MeSH
• polymerázová řetězová reakce MeSH
• pořadí genů MeSH
• rekombinace genetická MeSH
• replikon MeSH
• Salmonella typhimurium účinky léků   genetika   izolace a purifikace   patogenita   MeSH
• syntenie MeSH
• transpozibilní elementy DNA MeSH
• virulence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• DNA bakterií MeSH
• faktory virulence MeSH
• transpozibilní elementy DNA MeSH

Using transcriptional promoter fusions, we investigated the expression of selected SPI-1 and SPI-2 genes of Salmonella enterica serovar Typhimurium (S. Typhimurium). Promoters of genes related to the invasion of the epithelial cell (hilA, hilC, hilD, invF, sicA, sopA, sopB and sopE2) were active in Luria-Bertani (LB) medium and LB with butyrate but were suppressed by bile salts and in glucose minimal (M9) medium. Genes related to S. Typhimurium intracellular survival (phoP, ssrA, ssaB, ssaG, sifA, sifB and pipB) were characterized by their expression in stationary phase in LB and M9 medium. Activity of phoP and ssrA promoters indicated that these might be expressed inside the gut. SPI-1 genes were expressed on the transition to stationary phase while SPI-2 genes were expressed in stationary phase. Among SPI-1 genes, those with regulatory functions preceded in expression the effector genes and sop genes were expressed in the order of sopA, sopB and sopE2, showing hierarchy in the expression of S. Typhimurium virulence genes.

• MeSH
• bakteriální geny genetika   MeSH
• bakteriální proteiny metabolismus   MeSH
• časové faktory MeSH
• glukosa MeSH
• kultivační média MeSH
• promotorové oblasti (genetika) genetika   MeSH
• regulace genové exprese u bakterií * MeSH
• Salmonella typhimurium genetika   růst a vývoj   patogenita   MeSH
• salmonelóza mikrobiologie   MeSH
• trans-aktivátory metabolismus   MeSH
• virulence genetika   MeSH
• žlučové kyseliny a soli MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• glukosa MeSH
• HilA protein, Salmonella MeSH Prohlížeč
• kultivační média MeSH
• trans-aktivátory MeSH
• žlučové kyseliny a soli MeSH

Salmonella protease mutants, clpP and especially htrA, are candidate live oral vaccines in humans. A functional and mature immune system is, however, required to cope with them in mice. Here, we test the cytokine response of highly susceptible germ-free pigs to infection with Salmonella Typhimurium clpP and htrA mutants. Cytokine levels (IL-4, IL-10, IL-18 and IFN-gamma) were measured by ELISA in plasma and washes from the terminal small bowel 24h after oral challenge. Unlike the infection with the wild type strain, no IFN-gamma response and low IL-18 intestinal levels were found in pigs infected with the protease mutants. Despite this and regardless of partially reduced ability of htrA and clpP mutants to invade and multiply in a 3D4 porcine macrophage-like cell line, both the mutants were as virulent as was the wild type LT2 strain and caused fatal septicaemia in germ-free pigs. IFN-gamma and IL-18 response therefore did not correlate with the virulence of Salmonella Typhimurium. Our results indicate that htrA and clpP attenuations should be used with caution in populations in which an increased number of immunocompromised individuals can be expected.

• MeSH
• buněčné linie MeSH
• cytokiny imunologie   metabolismus   MeSH
• gnotobiologické modely MeSH
• makrofágy imunologie   mikrobiologie   MeSH
• mutace MeSH
• náchylnost k nemoci MeSH
• prasata MeSH
• proteasy genetika   metabolismus   MeSH
• Salmonella typhimurium enzymologie   genetika   imunologie   patogenita   MeSH
• salmonelová infekce u zvířat imunologie   metabolismus   mikrobiologie   MeSH
• salmonelové vakcíny imunologie   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny MeSH
• proteasy MeSH
• salmonelové vakcíny MeSH