Protozoan Cysts Act as a Survival Niche and Protective Shelter for Foodborne Pathogenic Bacteria

The production of cysts, an integral part of the life cycle of many free-living protozoa, allows these organisms to survive adverse environmental conditions. Given the prevalence of free-living protozoa in food-related environments, it is hypothesized that these organisms play an important yet currently underinvestigated role in the epidemiology of foodborne pathogenic bacteria. Intracystic bacterial survival is highly relevant, as this would allow bacteria to survive the stringent cleaning and disinfection measures applied in food-related environments. The present study shows that strains of widespread and important foodborne bacteria (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, and Listeria monocytogenes) survive inside cysts of the ubiquitous amoeba Acanthamoeba castellanii, even when exposed to either antibiotic treatment (100 μg/ml gentamicin) or highly acidic conditions (pH 0.2) and resume active growth in broth media following excystment. Strain- and species-specific differences in survival periods were observed, with Salmonella enterica surviving up to 3 weeks inside amoebal cysts. Up to 53% of the cysts were infected with pathogenic bacteria, which were located in the cyst cytosol. Our study suggests that the role of free-living protozoa and especially their cysts in the persistence and epidemiology of foodborne bacterial pathogens in food-related environments may be much more important than hitherto assumed.     

Deciphering the Acylation Pattern of Yersinia enterocolitica Lipid A

Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3′-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo₂-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of the LPS receptor by a LpxR-dependent deacylated LPS.     

Acanthamoeba castellanii Promotes the Survival of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a food-borne pathogen that naturally inhabits both marine and estuarine environments. Free-living protozoa exist in similar aquatic environments and function to control bacterial numbers by grazing on free-living bacteria. Protozoa also play an important role in the survival and spread of some pathogenic species of bacteria. We investigated the interaction between the protozoan Acanthamoeba castellanii and the bacterium Vibrio parahaemolyticus. We found that Acanthamoeba castellanii does not prey on Vibrio parahaemolyticus but instead secretes a factor that promotes the survival of Vibrio parahaemolyticus in coculture. These studies suggest that protozoa may provide a survival advantage to an extracellular pathogen in the environment.     

Influence of Native Microbiota on Survival of Ralstonia solanacearum Phylotype II in River Water Microcosms

Ralstonia solanacearum phylotype II biovar 2 causes bacterial wilt in solanaceous hosts, producing severe economic losses worldwide. Waterways can be major dissemination routes of this pathogen, which is able to survive for long periods in sterilized water. However, little is known about its survival in natural water when other microorganisms, such as bacteriophages, other bacteria, and protozoa, are present. This study looks into the fate of a Spanish strain of R. solanacearum inoculated in water microcosms from a Spanish river, containing different microbiota fractions, at 24°C and 14°C, for a month. At both temperatures, R. solanacearum densities remained constant at the initial levels in control microcosms of sterile river water while, by contrast, declines in the populations of the introduced strain were observed in the nonsterile microcosms. These decreases were less marked at 14°C. Lytic bacteriophages present in this river water were involved in the declines of the pathogen populations, but indigenous protozoa and bacteria also contributed to the reduced persistence in water. R. solanacearum variants displaying resistance to phage infection were observed, but only in microcosms without protozoa and native bacteria. In water microcosms, the temperature of 14°C was more favorable for the survival of this pathogen than 24°C, since biotic interactions were slower at the lower temperature. Similar trends were observed in microcosms inoculated with a Dutch strain. This is the first study demonstrating the influence of different fractions of water microorganisms on the survival of R. solanacearum phylotype II released into river water microcosms.     

Evaluation of a Modified Cefsulodin-Irgasan-Novobiocin Agar for Isolation of Yersinia spp

Y. enterocolitica and Y. pseudotuberculosis are important food borne pathogens. However, the presence of competitive microbiota makes the isolation of Y. enterocolitica and Y. pseudotuberculosis from naturally contaminated foods difficult. We attempted to evaluate the performance of a modified Cefsulodin-Irgasan-Novobiocin (CIN) agar in the differentiation of Y. enterocolitica from non-Yersinia species, particularly the natural intestinal microbiota. The modified CIN enabled the growth of Y. enterocolitica colonies with the same efficiency as CIN and Luria-Bertani agar. The detection limits of the modified CIN for Y. enterocolitica in culture medium (10 cfu/ml) and in artificially contaminated pork (10⁴ cfu/ml) were also comparable to those of CIN. However, the modified CIN provided a better discrimination of Yersinia colonies from other bacteria exhibiting Yersinia-like colonies on CIN (H₂S-producing Citrobacter freundii, C. braakii, Enterobacter cloacae, Aeromonas hydrophila, Providencia rettgeri, and Morganella morganii). The modified CIN exhibited a higher recovery rate of Y. enterocolitica from artificially prepared bacterial cultures and naturally contaminated samples compared with CIN. Our results thus demonstrated that the use of modified CIN may be a valuable means to increase the recovery rate of food borne Yersinia from natural samples, which are usually contaminated by multiple types of bacteria.     

Maximizing Plasmid Stability and Production of Released Proteins in Yersinia enterocolitica

Virulent serotypes of Yersinia enterocolitica carry a plasmid (pYV) encoding a family of proteins that are released into the medium and whose expression is temperature and calcium regulated. The plasmid is easily lost from cells during their growth in the laboratory. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with a monoclonal antibody (3.2C) that is specific for a 25-kDa released protein to show that 32°C is the lowest temperature at which plasmid-encoded proteins are expressed in quantity. The highest calcium concentration allowing full expression of these proteins was 445 to 545 μM at 32°C. Calcium concentrations of 745 μM and above at 37°C completely prevented the loss of pYV during multiple subcultures, while at 32°C, calcium concentrations of 245 μM and greater were sufficient to stabilize the plasmid. Growth of Y. enterocolitica at pH 5.5 was slower than at neutral pH values, but it also resulted in greatly increased stability of pYV. These studies showed that bacterial growth, retention of pYV, and expression of plasmid-encoded proteins may be maximized at 32°C with 445 μM calcium and that pYV stability is enhanced by growth at low pH. These observations suggest new approaches for isolation of plasmid-bearing virulent strains of Y. enterocolitica from samples contaminated with this organism and also may improve our understanding of pYV retention in vivo.     

Radiation Resistance and Injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25°C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and −30°C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at −20°C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at −20°C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at −20°C, nor did storage at −20°C alter the cell's resistance to irradiation at 25°C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36°C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36°C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5°C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36°C for 1 day than at 5°C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

Influence of Temperature and Pressure on the Lethality of Ultrasound

A specially designed resistometer was constructed, and the lethal effect on Yersinia enterocolitica of ultrasonic waves (UW) at different static pressures (manosonication [MS]) and of combined heat-UW under pressure treatments (manothermosonication [MTS]) was investigated. During MS treatments at 30°C and 200 kPa, the increase in the amplitude of UW of 20 kHz from 21 to 150 μm exponentially decreased decimal reduction time values (D_MS) from 4 to 0.37 min. When pressure was increased from 0 to 600 kPa at a constant amplitude (150 μm) and temperature (30°C), D_MS values decreased from 1.52 to 0.20 min. The magnitude of this decrease in D_MS declined progressively as pressure was increased. The influence of pressure on D_MS values was greater with increased amplitude of UW. Pressure alone of as much as 600 kPa did not influence the heat resistance of Y. enterocolitica (D₆₀ = 0.094; z = 5.65). At temperatures of as much as 58°C, the lethality of UW under pressure was greater than that of heat treatment alone at the same temperature. At higher temperatures, this difference disappeared. Heat and UW under pressure seemed to act independently. The lethality of MTS treatments appeared to result from the added effects of UW under pressure and the lethal effect of heat. The individual contributions of heat and of UW under pressure to the total lethal effect of MTS depended on temperature. The inactivating effect of UW was not due to titanium particles eroded from the sonication horn. The addition to the MS media of cysteamine did not increase the resistance of Y. enterocolitica to MS treatment. MS treatment caused cell disruption.     

Development of a Fluorogenic 5′ Nuclease PCR Assay for Detection of the ail Gene of Pathogenic Yersinia enterocolitica

In this report we describe the development and evaluation of a fluorogenic PCR assay for the detection of pathogenic Yersinia enterocolitica. The assay targets the chromosomally encoded attachment and invasion gene, ail. Three primer-probe sets (TM1, TM2, and TM3) amplifying different, yet overlapping, regions of ail were examined for their specificity and sensitivity. All three primer-probe sets were able to detect between 0.25 and 0.5 pg of purified Y. enterocolitica DNA. TM1 identified all 26 Y. enterocolitica strains examined. TM3 was able to detect all strains except one, whereas TM2 was unable to detect 10 of the Y. enterocolitica strains tested. None of the primer-probe sets cross-reacted with any of the 21 non-Y. enterocolitica strains examined. When the TM1 set was utilized, the fluorogenic PCR assay was able to detect ≤4 Y. enterocolitica CFU/ml in pure culture and 10 Y. enterocolitica CFU/ml independent of the presence of 10⁸ CFU of contaminating bacteria per ml. This set was also capable of detecting ≤1 CFU of Y. enterocolitica per g of ground pork or feces after a 24-h enrichment in a Yersinia selective broth.     

Growth of Listeria monocytogenes and Yersinia enterocolitica on Cooked Modified-Atmosphere-Packaged Poultry in the Presence and Absence of a Naturally Occurring Microbiota

Cooked poultry cuts were inoculated with five-strain composite mixtures of either Listeria monocytogenes or Yersinia enterocolitica (1,000 CFU/150-g piece), packaged in 44:56 CO₂-N₂, and stored at 3.5, 6.5, or 10°C for up to 5 weeks. Both L. monocytogenes and Y. enterocolitica grew under all test conditions. The presence of a naturally occurring microbiota did not influence the growth of either pathogen. Addition of lactate with the shelf life extender ALTA 2341 lengthened the lag phases of L. monocytogenes and Y. enterocolitica but did not prevent their growth.     

Identification of Yersinia enterocolitica using a random genomic DNA microarray chip

Aims: To fabricate a DNA chip containing random fragments of genomic DNA of Yersinia enterocolitica and to verify its diagnostic ability. Methods and Results: A DNA microarray chip was fabricated using randomly fragmented DNA of Y. enterocolitica. Chips were hybridized with genomic DNA extracted from other Y. enterocolitica strains, other Yersinia spp. and bacteria in different genera. Genomic DNA extracted from Y. enterocolitica showed a significantly higher hybridization rate compared with DNA of other Yersinia spp. or bacterial genera, thereby distinguishing it from other bacteria. Conclusions: A DNA chip containing randomly fragmented genomic DNA from Y. enterocolitica can detect Y. enterocolitica and clearly distinguish it from other Yersinia spp. and bacteria in different genera. Significance and Impact of the Study: A microarray chip containing randomly fragmented genomic DNA of Y. enterocolitica was fabricated without sequence information, and its diagnostic ability to identify Y. enterocolitica was verified.     

The complete genome sequence and comparative genome analysis of the high pathogenicity Yersinia enterocolitica strain 8081

The human enteropathogen, Yersinia enterocolitica, is a significant link in the range of Yersinia pathologies extending from mild gastroenteritis to bubonic plague. Comparison at the genomic level is a key step in our understanding of the genetic basis for this pathogenicity spectrum. Here we report the genome of Y. enterocolitica strain 8081 (serotype 0:8; biotype 1B) and extensive microarray data relating to the genetic diversity of the Y. enterocolitica species. Our analysis reveals that the genome of Y. enterocolitica strain 8081 is a patchwork of horizontally acquired genetic loci, including a plasticity zone of 199 kb containing an extraordinarily high density of virulence genes. Microarray analysis has provided insights into species-specific Y. enterocolitica gene functions and the intraspecies differences between the high, low, and nonpathogenic Y. enterocolitica biotypes. Through comparative genome sequence analysis we provide new information on the evolution of the Yersinia. We identify numerous loci that represent ancestral clusters of genes potentially important in enteric survival and pathogenesis, which have been lost or are in the process of being lost, in the other sequenced Yersinia lineages. Our analysis also highlights large metabolic operons in Y. enterocolitica that are absent in the related enteropathogen, Yersinia pseudotuberculosis, indicating major differences in niche and nutrients used within the mammalian gut. These include clusters directing, the production of hydrogenases, tetrathionate respiration, cobalamin synthesis, and propanediol utilisation. Along with ancestral gene clusters, the genome of Y. enterocolitica has revealed species-specific and enteropathogen-specific loci. This has provided important insights into the pathology of this bacterium and, more broadly, into the evolution of the genus. Moreover, wider investigations looking at the patterns of gene loss and gain in the Yersinia have highlighted common themes in the genome evolution of other human enteropathogens.     

Survival and Growth of Francisella tularensis in Acanthamoeba castellanii

Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis.     

Rapid species specific identification and subtyping of Yersinia enterocolitica by MALDI-TOF mass spectrometry

Yersinia enterocolitica are Gram-negative pathogens and known as important causes of foodborne infections. Rapid and reliable identification of strains of the species Y. enterocolitica within the genus Yersinia and the differentiation of the pathogenic from the non-pathogenic biotypes has become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid species identification and subtyping of Y. enterocolitica. To this end, we developed a reference MS database library including 19 Y. enterocolitica (non-pathogenic biotype 1A and pathogenic biotypes 2 and 4) as well as 24 non-Y. enterocolitica strains, belonging to eleven different other Yersinia spp. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2000 to 30,000 Da). Species-specific and biotype-specific biomarker protein mass patterns were determined for Y. enterocolitica. The defined biomarker mass patterns (SARAMIS SuperSpectrum™) were validated using 117 strains from various Y. enterocolitica bioserotypes in a blind-test. All strains were correctly identified and for all strains the mass spectrometry-based identification scheme yielded identical results compared to a characterization by a combination of biotyping and serotyping. Our study demonstrates that MALDI-TOF-MS is a reliable and powerful tool for the rapid identification of Y. enterocolitica strains to the species level and allows subtyping of strains to the biotype level.     

Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥10² CFU/ml in pure cultures and ≥10³ CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.     

Temperature-Dependent Galleria mellonella Mortality as a Result of Yersinia entomophaga Infection

The bacterium Yersinia entomophaga is pathogenic to a range of insect species, with death typically occurring within 2 to 5 days of ingestion. Per os challenge of larvae of the greater wax moth (Galleria mellonella) confirmed that Y. entomophaga was virulent when fed to larvae held at 25°C but was avirulent when fed to larvae maintained at 37°C. At 25°C, a dose of ∼4 × 10⁷ CFU per larva of a Y. entomophaga toxin complex (Yen-TC) deletion derivative, the Y. entomophaga ΔTC variant, resulted in 27% mortality. This low level of activity was restored to near-wild-type levels by augmentation of the diet with a sublethal dose of purified Yen-TC. Intrahemocoelic injection of ∼3 Y. entomophaga or Y. entomophaga ΔTC cells per larva gave a 4-day median lethal dose, with similar levels of mortality observed at both 25 and 37°C. Following intrahemocoelic injection of a Yen-TC YenA1 green fluorescent protein fusion strain into larvae maintained at 25°C, the bacteria did not fluoresce until the population density reached 2 × 10⁷ CFU ml⁻¹ of hemolymph. The observed cells also took an irregular form. When the larvae were maintained at 37°C, the cells were small and the observed fluorescence was sporadic and weak, being more consistent at a population density of ∼3 × 10⁹ CFU ml⁻¹ of hemolymph. These findings provide further understanding of the pathobiology of Y. entomophaga in insects, showing that the bacterium gains direct access to the hemocoelic cavity, from where it rapidly multiplies to cause disease.     

Outer Membrane Proteins Ail and OmpF of Yersinia pestis Are Involved in the Adsorption of T7-Related Bacteriophage Yep-phi

Yep-phi is a T7-related bacteriophage specific to Yersinia pestis, and it is routinely used in the identification of Y. pestis in China. Yep-phi infects Y. pestis grown at both 20°C and 37°C. It is inactive in other Yersinia species irrespective of the growth temperature. Based on phage adsorption, phage plaque formation, affinity chromatography, and Western blot assays, the outer membrane proteins of Y. pestis Ail and OmpF were identified to be involved, in addition to the rough lipopolysaccharide, in the adsorption of Yep-phi. The phage tail fiber protein specifically interacts with Ail and OmpF proteins, and residues 518N, 519N, and 523S of the phage tail fiber protein are essential for the interaction with OmpF, whereas residues 518N, 519N, 522C, and 523S are essential for the interaction with Ail. This is the first report to demonstrate that membrane-bound proteins are involved in the adsorption of a T7-related bacteriophage. The observations highlight the importance of the tail fiber protein in the evolution and function of various complex phage systems and provide insights into phage-bacterium interactions.     

Isolation ofYersinia enterocolitica andYersinia enterocolitica-like organisms from raw milk in Italy

Thirty samples of raw milk, originating from individual producers in the Turin area, were examined for the presence ofYersinia enterocolitica. A cold enrichment method with phosphate-buffered saline (PBS) 1/15M, pH 7.6, and sorbitol-bile-salts broth (SB) was used. After 7, 14, or 21 days at 4°–5°C, plating was performed on selective agar media directly (MacConkey agar andSalmonella-Shigella agar) after the alkali method was used. Six strains ofY. enterocolitica (biotype 1) and 32 strainsY. enterocolitica-like (threeY. fredericksenii; nineYersinia rhamnose-, melibiose+, -methyl-d-glucoside+, raffinose+, probablyYersinia intermedia biotype rhamnose-; and 20Y. intermedia) were isolated.Yersinia strains were found in 11 samples of raw milk, andY. enterocolitica in four samples.     

Dimethyl Adenosine Transferase (KsgA) Deficiency in Salmonella enterica Serovar Enteritidis Confers Susceptibility to High Osmolarity and Virulence Attenuation in Chickens

Dimethyl adenosine transferase (KsgA) performs diverse roles in bacteria, including ribosomal maturation and DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that a ksgA mutation in Salmonella enterica serovar Enteritidis results in impaired invasiveness in human and avian epithelial cells. In this study, we tested the virulence of a ksgA mutant (the ksgA::Tn5 mutant) of S. Enteritidis in orally challenged 1-day-old chickens. The ksgA::Tn5 mutant showed significantly reduced intestinal colonization and organ invasiveness in chickens compared to those of the wild-type (WT) parent. Phenotype microarray (PM) was employed to compare the ksgA::Tn5 mutant and its isogenic wild-type strain for 920 phenotypes at 28°C, 37°C, and 42°C. At chicken body temperature (42°C), the ksgA::Tn5 mutant showed significantly reduced respiratory activity with respect to a number of carbon, nitrogen, phosphate, sulfur, and peptide nitrogen nutrients. The greatest differences were observed in the osmolyte panel at concentrations of ≥6% NaCl at 37°C and 42°C. In contrast, no major differences were observed at 28°C. In independent growth assays, the ksgA::Tn5 mutant displayed a severe growth defect in high-osmolarity (6.5% NaCl) conditions in nutrient-rich (LB) and nutrient-limiting (M9 minimum salts) media at 42°C. Moreover, the ksgA::Tn5 mutant showed significantly reduced tolerance to oxidative stress, but its survival within macrophages was not impaired. Unlike Escherichia coli, the ksgA::Tn5 mutant did not display a cold-sensitivity phenotype; however, it showed resistance to kasugamycin and increased susceptibility to chloramphenicol. To the best of our knowledge, this is the first report showing the role of ksgA in S. Enteritidis virulence in chickens, tolerance to high osmolarity, and altered susceptibility to kasugamycin and chloramphenicol.     

CD8+ T Cells Restrict Yersinia pseudotuberculosis Infection: Bypass of Anti-Phagocytosis by Targeting Antigen-Presenting Cells

All Yersinia species target and bind to phagocytic cells, but uptake and destruction of bacteria are prevented by injection of anti-phagocytic Yop proteins into the host cell. Here we provide evidence that CD8⁺ T cells, which canonically eliminate intracellular pathogens, are important for restricting Yersinia, even though bacteria are primarily found in an extracellular locale during the course of disease. In a model of infection with attenuated Y. pseudotuberculosis, mice deficient for CD8⁺ T cells were more susceptible to infection than immunocompetent mice. Although exposure to attenuated Y. pseudotuberculosis generated T_H1-type antibody responses and conferred protection against challenge with fully virulent bacteria, depletion of CD8⁺ T cells during challenge severely compromised protective immunity. Strikingly, mice lacking the T cell effector molecule perforin also succumbed to Y. pseudotuberculosis infection. Given that the function of perforin is to kill antigen-presenting cells, we reasoned that cell death marks bacteria-associated host cells for internalization by neighboring phagocytes, thus allowing ingestion and clearance of the attached bacteria. Supportive of this model, cytolytic T cell killing of Y. pseudotuberculosis–associated host cells results in engulfment by neighboring phagocytes of both bacteria and target cells, bypassing anti-phagocytosis. Our findings are consistent with a novel function for cell-mediated immune responses protecting against extracellular pathogens like Yersinia: perforin and CD8⁺ T cells are critical for hosts to overcome the anti-phagocytic action of Yops.