The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host.

Francisella tularensis is the causative agent of tularemia and a category A potential agent of bioterrorism, but the pathogenic mechanisms of F. tularensis are largely unknown. Our previous transposon mutagenesis screen identified 95 lung infectivity-associated F. tularensis genes, including those encoding the Lon and ClpP proteases. The present study validates the importance of Lon and ClpP in intramacrophage growth and infection of the mammalian host by using unmarked deletion mutants of the F. tularensis live vaccine strain (LVS). Further experiments revealed that lon and clpP are also required for F. tularensis tolerance to stressful conditions. A quantitative proteomic comparison between heat-stressed LVS and the isogenic Lon-deficient mutant identified 29 putative Lon substrate proteins. The follow-up protein degradation experiments identified five substrates of the F. tularensis Lon protease (FTL578, FTL663, FTL1217, FTL1228, and FTL1957). FTL578 (ornithine cyclodeaminase), FTL663 (heat shock protein), and FTL1228 (iron-sulfur activator complex subunit SufD) have been previously described as virulence-associated factors in F. tularensis Identification of these Lon substrates has thus provided important clues for further understanding of the F. tularensis stress response and pathogenesis. The high-throughput approach developed in this study can be used for systematic identification of the Lon substrates in other prokaryotic and eukaryotic organisms.

Biosafety level 2 model of pneumonic plague and protection studies with F1 and Psa.

Attenuated Yersinia pestis pgm strains, such as KIM5, lack the siderophore yersiniabactin. Strain KIM5 does not induce significant pneumonia when delivered intranasally. In this study, mice were found to develop pneumonia after intranasal challenge with strain KIM5 when they were injected intraperitoneally with iron dextran, though not with iron sulfate. KIM5-infected mice treated daily with 4 mg iron dextran died in 3 days with severe pneumonia. Pneumonia was less severe if 4 mg iron dextran was administered only once before infection. The best-studied experimental vaccine against plague currently consists of the Yersinia pestis capsular antigen F1 and the type 3 secreted protein LcrV. The F1 antigen was shown to be protective against KIM5 infections in mice administered iron dextran doses leading to light or severe pneumonia, supporting the use of an iron dextran-treated model of pneumonic plague. Since F1 has been reported to be incompletely protective in some primates, and bacterial isolates lacking F1 are still virulent, there has been considerable interest in identifying additional protective subunit immunogens. Here we showed that the highly conserved Psa fimbriae of Y. pestis (also called pH 6 antigen) are expressed in murine organs after infection through the respiratory tract. Studies with iron dextran-treated mice showed that vaccination with the Psa fimbrial protein together with an adjuvant afforded incomplete but significant protection in the mouse model described. Therefore, further investigations to fully characterize the protective properties of the Psa fimbriae are warranted.

Outer membrane protein A (OmpA) of Cronobacter sakazakii binds fibronectin and contributes to invasion of human brain microvascular endothelial cells.

Cronobacter sakazakii is an emerging foodborne pathogen that causes severe meningitis and meningoencephalitis in neonates. Currently there is a dearth of information available on the virulence factors of C. sakazakii and the pathogenic mechanisms involved in its neonatal infections. The invasion and translocation of the blood-brain barrier formed by brain microvascular endothelial cells (BMEC) is critical in the pathogenesis of neonatal bacterial meningitis. Because bacterial binding of fibronectin is an initial step in the invasion of BMEC, the role of a major surface-expressed fibronectin-binding protein of C. sakazakii in invasion of BMEC was investigated. Outer membrane protein A was identified as a major fibronectin-binding protein of C. sakazakii, and an isogenic ompA mutant of C. sakazakii exhibited significantly (p < 0.05) attenuated invasion in BMEC compared with the wild-type strain. The findings of this study indicate that outer membrane protein A is one of the determinants that contribute to C. sakazakii invasion of human BMEC in vitro, and may potentially play a role in the pathogenesis of neonatal meningitis caused by this organism.

Inactivation of Listeria monocytogenes on frankfurters by monocaprylin alone or in combination with acetic acid.

The antilisterial activity of monocaprylin (MC) and its combination with acetic acid (AA) on frankfurters was investigated. Each frankfurter was surface inoculated with a three-strain mixture of Listeria monocytogenes to obtain an inoculation level of 4.0 log CFU per frankfurter, and then dipped for 35 s in sterile deionized water (45 or 50 degrees C) containing 1% ethanol (control), 50 mM MC plus 1% ethanol, 1% AA plus 1% ethanol, or 50 mM MC plus 1% AA plus 1% ethanol. Samples were vacuum packaged, stored at 4 degrees C for 77 days, and analyzed for L. monocytogenes. Sensory odor and color of frankfurters were evaluated using a 9-point hedonic scale. Color was also objectively measured using the Minolta Chroma Meter. From day 0 to day 77, population counts of L. monocytogenes on frankfurters dipped in antimicrobial solutions at 50 degrees C were consistently lower than the control counts. Similar results were observed for samples treated at 45 degrees C. However, L. monocytogenes grew readily on control samples at both temperatures. Dipping of frankfurters in antimicrobial solutions (45 or 50 degrees C) significantly reduced (P < 0.05) the populations of L. monocytogenes. After 70 days of storage, L. monocytogenes was completely killed in samples dipped in MC+AA solution at 50 degrees C. The antimicrobial treatments did not affect the odor or color of the samples (P > 0.05). Overall, results indicated that dipping of frankfurters with MC reduced L. monocytogenes, and inclusion of AA further enhanced MC antilisterial activity, without any negative effect on odor or color.

Outer membrane vesicle-associated lipase FtlA enhances cellular invasion and virulence in Francisella tularensis LVS.

Francisella tularensis is a highly infectious intracellular pathogen that infects a wide range of host species and causes fatal pneumonic tularemia in humans. ftlA was identified as a potential virulence determinant of the F. tularensis live vaccine strain (LVS) in our previous transposon screen, but its function remained undefined. Here, we show that an unmarked deletion mutant of ftlA was avirulent in a pneumonia mouse model with a severely impaired capacity to infect host cells. Consistent with its sequence homology with GDSL lipase/esterase family proteins, the FtlA protein displayed lipolytic activity in both E. coli and F. tularensis with a preference for relatively short carbon-chain substrates. FtlA thus represents the first F. tularensis lipase to promote bacterial infection of host cells and in vivo fitness. As a cytoplasmic protein, we found that FtlA was secreted into the extracellular environment as a component of outer membrane vesicles (OMVs). Further confocal microscopy analysis revealed that the FtlA-containing OMVs isolated from F. tularensis LVS attached to the host cell membrane. Finally, the OMV-associated FtlA protein complemented the genetic deficiency of the ΔftlA mutant in terms of host cell infection when OMVs purified from the parent strain were co-incubated with the mutant bacteria. These lines of evidence strongly suggest that the FtlA lipase promotes F. tularensis adhesion and internalization by modifying bacterial and/or host molecule(s) when it is secreted as a component of OMVs.

Antibacterial effect of monocaprylin on Escherichia coli O157:H7 in apple juice.

The antibacterial effect of low concentrations of monocaprylin on Escherichia coli O157:H7 in apple juice was investigated. Apple juice alone (control) or containing 2.5 mM (0.055%) or 5 mM monocaprylin was inoculated with a five-strain mixture of E. coli O157:H7 at approximately 6.0 log CFU/ml. The juice samples were stored at 23 or 4 degrees C for 14 or 21 days, respectively, and the population of E. coli O157:H7 was determined on tryptic soy agar plates supplemented with 0.6% yeast extract. At both storage temperatures, the population of E. coli O157:H7 in monocaprylin-supplemented juice samples was significantly lower (P < 0.05) than that in the control samples. The concentration of monocaprylin and the storage temperature had a significant effect on the inactivation of E. coli O157:H7 in apple juice. Monocaprylin at 5 mM was significantly more effective than 2.5 mM monocaprylin for killing E. coli O157:H7 in apple juice. Inactivation of E. coli O157:H7 by monocaprylin was more pronounced in juice stored at 23 degrees C than in the refrigerated samples. Results of this study indicated that monocaprylin is effective for killing E. coli O157:H7 in apple juice, but detailed sensory studies are needed to determine the organoleptic properties of apple juice containing monocaprylin.

Survival and growth characteristics of Escherichia coli O157:H7 in pasteurized and unpasteurized Cheddar cheese whey.

The objective of this study was to determine the survival and growth characteristics of Escherichia coli O157:H7 in whey. A five-strain mixture of E. coli O157:H7 was inoculated into 100 ml of fresh, pasteurized or unpasteurized Cheddar cheese whey (pH 5.5) at 10(5) or 10(2) CFU/ml, and stored at 4, 10 or 15 degrees C. The population of E. coli O157:H7 (on Sorbitol MacConkey agar supplemented with 0.1% 4-methylumbelliferyl-beta-D-glucuronide) and lactic acid bacteria (on All Purpose Tween agar) were determined on days 0, 1, 4, 7, 14, 21 and 28. At all storage temperatures, survival of E. coli O157:H7 was significantly higher (P<0.01) in the pasteurized whey compared to that in the unpasteurized samples. At 10 and 15 degrees C, E. coli O157:H7 in pasteurized whey significantly (P<0.05) increased during the first week of storage, followed by a decrease thereafter. However at the same temperatures, E. coli O157:H7 exhibited a steady decline in the unpasteurized samples from day 0. At 4 degrees C, E. coli O157:H7 did not grow in pasteurized and unpasteurized whey; however, the pathogen persisted longer in pasteurized samples. At all the three storage temperatures, E. coli O157:H7 survived up to day 21 in the pasteurized and unpasteurized whey. The initial load of lactic acid bacteria in the unpasteurized whey samples was approximately 7.0 log10 CFU/ml and, by day 28, greater than 3.0 log10 CFU/ml of lactic acid bacteria survived in unpasteurized whey at all temperatures, with the highest counts recovered at 4 degrees C. Results indicate the potential risk of persistence of E. coli O157:H7 in whey in the event of contamination with this pathogen.

Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation.

Staphylococcus aureus is one of the most common pathogens responsible for contagious mastitis in ruminants. The ability of S. aureus to form biofilm in vivo is considered to be a major virulence factor influencing its pathogenesis in mastitis. The objectives of the study were to examine in vitro slime production, biofilm formation, and the presence of the ica gene locus and icaA and icaD genes in S. aureus isolates from bovine mastitis. Thirty-two of the 35 isolates tested produced slime on Congo red agar, whereas only 24 of the isolates were found to produce biofilm in vitro. However, all the 35 isolates possessed the ica locus, icaA and icaD genes. This study indicates a high prevalence of the ica genes among S. aureus mastitis isolates, and their presence is not always associated with in vitro formation of slime or biofilm. A combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in S. aureus.

Inactivation of Enterobacter sakazakii in reconstituted infant formula by monocaprylin.

Enterobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in neonates and children, with a mortality rate of 14%. Epidemiological studies have implicated dried infant formula as the principal source of the pathogen. Caprylic acid is a natural eight-carbon fatty acid present in breast milk and bovine milk and is approved as generally recognizable as safe by the U.S. Food and Drug Administration. The objective of this study was to determine the antibacterial effect of monocaprylin (monoglyceride ester of caprylic acid) on E. sakazakii in reconstituted infant formula. A five-strain mixture of E. sakazakii was inoculated into 10-ml samples of reconstituted infant formula (at 6.0 log CFU/ml) followed by 0, 25, or 50 mM (1%) monocaprylin. The samples were incubated at 37 or 23 degrees C for 0, 1, 6, and 24 h and at 8 or 4 degrees C for 0, 6, 24, and 48 h, and the surviving populations of E. sakazakii at each sampling time were counted. The treatments containing monocaprylin significantly reduced the population of E. sakazakii (P < 0.05) compared with the controls. Monocaprylin (50 mM) reduced the pathogen by >5 log CFU/ml by 1 h of incubation at 37 or 23 degrees C and by 24 h of incubation at 8 or 4 degrees C. Results indicate that monocaprylin could potentially be used to inactivate E. sakazakii in reconstituted infant formula; however, sensory studies are warranted before its use can be recommended.

The capBCA Locus is Required for Intracellular Growth of Francisella tularensis LVS.

Francisella tularensis is the causative agent of tularemia and a category A bioterrorism agent. The molecular basis for the extreme virulence of F. tularensis remains unclear. Our recent study found that capBCA, three neighboring genes, are necessary for the infection of F. tularensis live vaccine strain (LVS) in a respiratory infection mouse model. We here show that the capBCA genes are necessary for in vivo growth of F. tularensis LVS in the lungs, spleens, and livers of BALB/c mice. Unmarked deletion of capBCA in type A strain Schu S4 resulted in significant attenuation in virulence although the level of the attenuation in Schu S4 was much less profound than in LVS. We further demonstrated that CapB protein is produced at a low level under the in vitro culture conditions, and capB alone is necessary for in vivo growth of F. tularensis LVS in the lungs of BALB/c mice. Finally, deletional mutations in capB alone or capBCA significantly impaired intracellular growth of F. tularensis LVS in cultured macrophages, thus suggesting that the capBCA genes are necessary for intracellular adaptation of F. tularensis. The requirement of this gene locus in intracellular adaption at least in part explains the significant attenuation of F. tularensis capBCA mutants in virulence.