Analyzing Thermal Stability of Cell Membrane of Salmonella Using Time-Multiplexed Impedance Sensing

Heat treatment is one of the most widely used methods for inactivation of bacteria in food products. Heat-induced loss of bacterial viability has been variously attributed to protein denaturation, oxidative stress, or membrane leakage; indeed, it is likely to involve a combination of these processes. We examine the effect of mild heat stress (50–55°C for ≤12 min) on cell permeability by directly measuring the electrical conductance of samples of Salmonella enterica serovar Typhimurium to answer a fundamental biophysical question, namely, how bacteria die under mild heat stress. Our results show that when exposed to heat shock, the cell membrane is damaged and cells die mainly due to the leakage of small cytoplasmic species to the surrounding media without lysis (confirmed by fluorescent imaging). We measured the conductance change, ΔY, of wild-type versus genetically modified heat-resistant (HR) cells in response to pulse and ramp heating profiles with different thermal time constants. In addition, we developed a phenomenological model to correlate the membrane damage, cytoplasmic leakage, and cell viability. This model traces the differential viability and ΔY of wild-type and HR cells to the difference in the effective activation energies needed to permeabilize the cells, implying that HR cells are characterized by stronger lateral interactions between molecules, such as lipids, in their cell envelope.     

An epidemic of plasmids? Dissemination of extended-spectrum cephalosporinases among Salmonella and other Enterobacteriaceae

CTX-M- and AmpC-type beta-lactamases comprise the two most rapidly growing populations among the extended-spectrum cephalosporinases. The evolution and dissemination of resistance genes encoding these enzymes occur mostly through the transmission of plasmids. The high prevalence of clinical isolates of Enterobacteriaceae producing the plasmid-mediated extended-spectrum cephalosporinases resembles an epidemic of plasmids, and has generated serious therapeutic problems. This review describes the emergence and worldwide spread of various classes of plasmid-mediated extended-spectrum cephalosporinases in Salmonella and other Enterobacteriaceae, the transfer mechanism of the plasmids, detection methods, and therapeutic choices.     

Febrile Gastroenteritis Due to Salmonella thompson—Report of an Outbreak

Salmonella thompson, a common pathogen of poultry, has received scant attention as a cause of human gastroenteritis. At least 45 persons were infected with S thompson in Sacramento, California, after eating at a chicken restaurant and 38 became symptomatic. Ten required admission to hospital, and all were treated with antibiotics and improved. In 19 cases cultures of stool specimens for S thompson over a 60-day period showed slower but statistically insignificant differences in salmonellal elimination in 7 patients who received antibiotics when compared with 12 who were untreated. We report this outbreak to increase awareness of the virulence and prevalence of gastroenteritis due to S thompson.     

Selective Inhibition of Escherichia coli in the Presence of Salmonella typhimurium by Phenethyl β-D-Galactopyranoside

Phenethyl β-d-galactopyranoside (PEG) was hydrolyzed by the β-galactosidase of Escherichia coli to form the toxic product phenethyl alcohol. Salmonella typhimurium did not hydrolyze PEG. In mixed culture, the ratio of S. typhimurium to E. coli was increased by growing the organisms in lactose broth containing 2.5% PEG. The high concentration of PEG required for inhibition of E. coli can be attributed to inadequate cell permeability rather than to prevention of β-galactosidase induction.     

Azidoalanine mutagenicity in Salmonella: Effect of homologation and α-methyl substitution

Azide mutagenicity in susceptible non-mammalian systems involves the requisite formation of l-azidoalanine, a novel mutagenic amino acid. The biochemical mechanism(s) of azidoalanine-induced mutagenesis, however, is not known. Previous studies of the structural requirements for azidoalanine mutagenicity suggested the importance of free l-amino acid character, and that bioactivation of azidoalanine to the ultimate mutagenic species is required. To gain more insight into possible enzymatic processing, the α-methyl analogue, α-methylazidialanine, and the homologue, 2-amino-4-azidobutonoic acid, were synthesized and tested for mutagenic potency in Salmonella typhimurium strain TA1530. In addition, azidoacetic acid, a possible azidoalanine metabolite, was prepared and tested. The results show that α-methyl substitution effectively blocks the mutagenic effects of azidoalanine with α-methyl-azidoalanine being nearly devoid of mutagenic activity. In contrast, homologation of azidoalanine to yield 2-amino-4-azidobutanoic acid produces a marked increase in molar mutagenic potency. As with azidoalanine, the mutagenic activity of this homologue is associated with the l-isomer. Azidoacetic acid, however, was only very weakly mutagenic when tested as either the free acid or ethyl ester. This low mutagenic potency may indicate that bioactivation does not involve the entry of azide-containing azidoalanine catabolite into the Kreb's cycle. The high potency of 2-amino-4-azidobutanoic acid may be indicative of more efficient bioactivation and/or greater intrinsic activity. Importantly, the latter finding clearly shows that potent azido-amino acid mutagenicity is not limited to azidoalanine alone.     

The frequency and duration of Salmonella–macrophage adhesion events determines infection efficiency

Salmonella enterica causes a range of important diseases in humans and a in a variety of animal species. The ability of bacteria to adhere to, invade and survive within host cells plays an important role in the pathogenesis of Salmonella infections. In systemic salmonellosis, macrophages constitute a niche for the proliferation of bacteria within the host organism. Salmonella enterica serovar Typhimurium is flagellated and the frequency with which this bacterium collides with a cell is important for infection efficiency. We investigated how bacterial motility affects infection efficiency, using a combination of population-level macrophage infection experiments and direct imaging of single-cell infection events, comparing wild-type and motility mutants. Non-motile and aflagellate bacterial strains, in contrast to wild-type bacteria, collide less frequently with macrophages, are in contact with the cell for less time and infect less frequently. Run-biased Salmonella also collide less frequently with macrophages but maintain contact with macrophages for a longer period of time than wild-type strains and infect the cells more readily. Our results suggest that uptake of S. Typhimurium by macrophages is dependent upon the duration of contact time of the bacterium with the cell, in addition to the frequency with which the bacteria collide with the cell.     

X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of d-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 Å resolution. The active site of the enzyme contains l-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme.     

Do Salmonella carry spare tyres?

Salmonellae are enterobacteria that have the unique ability to change their flagellar composition by switching expression among two loci that encode the major flagellin protein. This property is not available to all Salmonella, but is species, subspecies and serotype specific. Curiously, the subsequent loss of the second locus in some lineages of Salmonella has apparently been tolerated and, indeed, has led to considerable success for some lineages. We discuss here an evolutionary model for maintenance of this unique function and the possible evolutionary advantages of loss or preservation of this mechanism. We hypothesize that the second flagellin locus is a genetic 'spare tyre' used in particular environmental circumstances.     

Spontaneous Excision of the Salmonella enterica Serovar Enteritidis-Specific Defective Prophage-Like Element φSE14

Salmonella enterica serovar Enteritidis has emerged as a major health problem worldwide in the last few decades. DNA loci unique to S. Enteritidis can provide markers for detection of this pathogen and may reveal pathogenic mechanisms restricted to this serovar. An in silico comparison of 16 Salmonella genomic sequences revealed the presence of an ∼12.5-kb genomic island (GEI) specific to the sequenced S. Enteritidis strain NCTC13349. The GEI is inserted at the 5′ end of gene ydaO (SEN1377), is flanked by 308-bp imperfect direct repeats (attL and attR), and includes 21 open reading frames (SEN1378 to SEN1398), encoding primarily phage-related proteins. Accordingly, this GEI has been annotated as the defective prophage SE14 in the genome of strain NCTC13349. The genetic structure and location of φSE14 are conserved in 99 of 103 wild-type strains of S. Enteritidis studied here, including reference strains NCTC13349 and LK5. Notably, an extrachromosomal circular form of φSE14 was detected in every strain carrying this island. The presence of attP sites in the circular forms detected in NCTC13349 and LK5 was confirmed. In addition, we observed spontaneous loss of a tetRA-tagged version of φSE14, leaving an empty attB site in the genome of strain NCTC13349. Collectively, these results demonstrate that φSE14 is an unstable genetic element that undergoes spontaneous excision under standard growth conditions. An internal fragment of φSE14 designated Sdf I has been used as a serovar-specific genetic marker in PCR-based detection systems and as a tool to determine S. Enteritidis levels in experimental infections. The instability of this region may require a reassessment of its suitability for such applications.The genus Salmonella comprises a heterogeneous group of Gram-negative bacteria, differentiable by biochemical and serological properties. More than 2,500 Salmonella serovars have been identified according to the serospecificities of the somatic and flagellar antigens. Some serovars, exemplified by Salmonella enterica serovar Typhimurium and S. Enteritidis, can infect a broad range of hosts. However, a subset of serovars, such as S. Typhi, a human-specific pathogen, show a high degree of adaptation to a specific host.In the last few decades, S. Enteritidis has emerged as a major health problem worldwide (31). This pathogen colonizes the reproductive organs of infected birds without causing discernible illness and survives host defenses during the formation of the egg (25, 27). The production of a capsule-like O antigen structure by certain wild-type strains of S. Enteritidis (30, 46) has been associated with reproductive tract tropism and improved survival within eggs (26, 27, 45). Egg contamination can originate before oviposition by direct contamination of the yolk, albumen, or eggshell membranes with bacteria from the infected reproductive organs of the birds or after or during oviposition by penetration of bacteria from contaminated feces through the eggshell (8, 14, 25). Transmission of the bacterium to humans occurs mainly through the consumption of contaminated eggs or egg products (8, 14, 25). Upon infection of a human host, S. Enteritidis causes self-limiting gastroenteritis similar to that caused by other nontyphoidal Salmonella serovars.According to information gathered from 84 countries responding to a global survey conducted by the World Health Organization (WHO), S. Enteritidis and S. Typhimurium accounted for ∼70% of all human and nonhuman isolates of Salmonella reported worldwide between 1995 and 2008. In fact, S. Enteritidis alone accounted for 61.4% of the ∼1.5 million human isolates of Salmonella reported during this period, according to the WHO Global Foodborne Infections Network Country Databank (http://www.who.int/salmsurv). Remarkably, S. Enteritidis is the second most prevalent cause of Salmonella infection in humans, after S. Typhimurium, in the United States (10).The high global prevalence of S. Enteritidis makes the development of a rapid, sensitive, and highly specific detection system critical to collect accurate epidemiologic data. The identification of loci that serve as specific markers for DNA-based identification of this pathogen may also provide insights into pathogenic mechanisms restricted to this serovar. Genomic regions that are unique to given serovars are especially suitable for such epidemiologic detection (3). For instance, Agron and colleagues identified an S. Enteritidis-specific genomic region of ∼4,060 bp adjacent to the ydaO gene, carrying six open reading frames (ORFs) that they designated lygA to lygF (1). A PCR-based assay successfully detected the presence of an internal fragment of this serovar-specific region in most strains in a diverse collection of clinical and environmental S. Enteritidis isolates and not in 73 non-Enteritidis isolates of S. enterica representing 34 different serovars (1). Since then, this region has been widely used as an S. Enteritidis-specific molecular marker in the development of several PCR-based assays for detection and epidemiological typing of Salmonella serovars in clinical and environmental samples (2, 11, 32, 37, 44, 53). Recently, an S. Enteritidis-specific real-time quantitative PCR (qPCR) assay based on the detection of this region was developed (15). This qPCR assay has been used in a series of studies of the distribution and replication kinetics of S. Enteritidis in experimentally infected animals (16-21).We performed a bioinformatic study to identify genomic regions specific to S. Enteritidis and found a genomic island (GEI) that includes the S. Enteritidis-specific locus lyg (1). This island has been annotated as the defective prophage SE14 in the genome of S. Enteritidis strain NCTC13349 (52). Although we demonstrate that the location in the genome and the overall genetic structure of the island are conserved in wild-type isolates of S. Enteritidis from different origins, we detected strains that do not carry the island in their genomes. Finally, we demonstrate here that the island corresponds to an unstable element that undergoes spontaneous excision from the genome of S. Enteritidis under standard growth conditions.     

An Epidemic of Salmonella Infantis Infection in Finnish Broiler Chickens in 1975–76

A large S. infantis infection epidemic in broiler chickens was studied during a period of one year. The outbreak affected three broiler producing companies in Finland. The infection spread to breeding farms according to available data during the summer of 1975. The epidemic still prevailed at the end of the studies on the farms of certain companies. The origin of the infection and the means of its spreading could not be ascertained. Some epidemiological evidence suggesting that a hatchery might have spread the infection was found. Contaminated feed may also have been involved, although the findings do not support feed as the principal vehicle in the epidemic. A complex pattern of transmission is most probable. A microbiological preventive method based on the feeding of a culture of intestinal flora of adult chickens to newly hatched broiler chickens was used on many farms in the study. The feeding of the culture lowered the proportion of infected flocks on the farms and significantly lowered the number of infected birds in those flocks, where the prevention was not complete. kw|Keywords|k]Salmonella infantis; k]infection; k]epidemic; k]broiler chicken     

Adsorption of phage λ to Salmonella typhimurium lamB + requires the presence of lipopolysaccharide in the outer membrane

Summary Two S. typhimurium strains TA1534 (rfa ⁺) and TA1538 (rfaE) were transformed with the lamB expression plasmid pAMH70. Transposition events with placMu55 hybrid phage were successful only with TA1534/pAMH70 strain. Using SDS-PAGE, the LamB protein was present in the total cell proteins but not in the outer membrane proteins of the TA1538/pAMH70 strain. The LamB protein must linked to the LPS of the outer membrane to allow adsorption of phage in S. typhimurium.     

Regulation of septation: a novel role for SerC/PdxF in Salmonella?

The sfiW locus of Salmonella enterica, previously identified by mutations that suppress the cell division defect of His-constitutive (His(c)) strains, corresponds to serC, the bifunctional gene for phosphoserine-oxoglutarate aminotransferase (SerC) and 2-ketoerythroic acid 4-phosphate transaminase (PdxF). SerC- mutants form small, nearly spherical cells in a wild-type (His+) background, suggesting that the SerC/PdxF product acts as a septation antagonist. Suppression of His(c) filamentation by serC mutations may be explained by loss of the anti-septation activity of SerC/PdxF. The isolation of serC alleles that have lost their biosynthetic activities but are still able to inhibit septum formation suggests that the anti-septation activity of the SerC/PdxF product is unrelated to its known roles in serine and pyridoxine biosynthesis.