Response of Listeria monocytogenes to Disinfection Stress at the Single-Cell and Population Levels as Monitored by Intracellular pH Measurements and Viable-Cell Counts

Listeria monocytogenes has a remarkable ability to survive and persist in food production environments. The purpose of the present study was to determine if cells in a population of L. monocytogenes differ in sensitivity to disinfection agents as this could be a factor explaining persistence of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level by measuring intracellular pH (pH_i) over time by fluorescence ratio imaging microscopy. pH_i values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pH_i was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pH_i 5 to 5.5) (P < 0.05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (P < 0.05). NaCl protected attached cells through drying but not during disinfection. This study indicates that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present.Listeria monocytogenes is a food-borne, human pathogen that has a remarkable ability to colonize food-processing environments (5, 16, 20, 21, 26, 29). Some L. monocytogenes strains can persist for years in food-processing plants (11, 14, 20, 27), and specific molecular subtypes can repeatedly be isolated from the processing environment (29) despite being very infrequent in the outdoor environment (9). This ability to persist has, hitherto, not been linked to any specific genetic or phenotypic trait.It has been suggested that persistent L. monocytogenes strains may be more tolerant or resistant to cleaning and especially disinfectants used in the food industry. Aase et al. (1) found increased tolerance to both benzalkonium chloride and ethidium bromide in L. monocytogenes isolates that had persisted for more than 4 years; however, other studies have not been able to link persistence and tolerance to disinfectants (6, 10, 11, 13). We recently compared disinfection sensitivities of persistent and presumed nonpersistent L. monocytogenes strains using viable-cell counts and did not find the latter group more sensitive to the two disinfectants Triquart SUPER and Incimaxx DES than persistent strains (13). However, we found that for all subtypes of L. monocytogenes, growth with NaCl increased the tolerance of planktonic L. monocytogenes cells to Incimaxx DES, whereas spot-inoculated, dried L. monocytogenes cells were not protected by NaCl against disinfection.There is no doubt that L. monocytogenes will be completely inactivated at the disinfectant concentrations recommended for use in the food industry; however, the efficiency of the disinfectant is very much influenced by the presence of organic material being inactivated by the presence of food debris. Hence, it is likely that the bacterial cell in a food production environment may be exposed to concentrations at a sublethal level. It is currently not known if treatment with a sublethal concentration of disinfectant affects the entire bacterial population or only attacks a fraction of the cell population, leaving another fraction of cells unaffected. In case of the latter, some bacterial cells may be able to survive the disinfection treatment. The potential presence of such tolerant subpopulations could, ultimately, ensure that the genome is propagated, leading to persistence.The presence of a more tolerant subpopulation can be determined on the single-cell level. Flow cytometry is a rapid method useable for measurements at the single-cell resolution (22); however, it cannot monitor the same single cells over time. Optical microscopy combined with microfluidic devices that allow measurement of growth of single cells is a useful technique (2), and in situ analyses of the physiological condition of single cells by the fluorescence ratio imaging microscopy (FRIM) technique represents another elegant approach (25). FRIM enables studies of dynamic changes with high sensitivity and on the single-cell level in important physiological parameters: e.g., intracellular pH (pH_i). Listeria maintains its pH_i within a narrow range of 7.6 to 8 at extracellular pH (pH_ex) values of 5.0 to 8.0 (4, 25) and at pH_ex 4.0 with the presence of glucose (23). It is believed that viable cells need to maintain a transmembrane pH gradient with their pH_i above the pH_ex, and failure to maintain pH_i homeostasis indicates that the bacterial cell is severely stressed and ultimately leads to loss of cell viability. FRIM has been used to determine the pH_i of L. monocytogenes after exposure to osmotic and acid stress (7, 23). Also, the dissipation of the pH gradient in L. monocytogenes after exposure to different bacteriocins has been determined with FRIM (4, 12). Hornbæk et al. (12) found that treatment with subinhibitory concentrations of leucocin and nisin gave rise to two subpopulations: one consisting of cells with a dissipated pH gradient (ΔpH) and the other consisting of cells that maintained ΔpH, which could indicate phenotypic heterogeneity.The aim of the present study was to investigate the physiological effects of the disinfectant Incimaxx DES at sublethal and lethal concentrations on single cells and the population level of a persistent L. monocytogenes strain to study a possible subdivision of sensitivity in the population. We also addressed the potential protective effect of NaCl against disinfection and compared sensitivities in a population of planktonic and attached bacteria. We applied the in situ technique FRIM and compared the pH_i measurements with the traditional viable-cell-count method.(Part of the results have been presented at a poster session at the 95th International Association for Food Protection annual meeting in Columbus, OH, 3 to 6 August 2008.)     

Telomere Biology and Cellular Aging in Nonhuman Primate Cells

To determine how cellular aging is conserved among primates, we analyzed the replicative potential and telomere shortening in skin fibroblasts of anthropoids and prosimians. The average telomere length of the New World primates Ateles geoffroyi (spider monkey) and Saimiri sciureus (squirrel monkey) and the Old World primates Macaca mulatta (rhesus monkey), Pongo pygmaeus (orangutan), and Pan paniscus (pigmy chimpanzee) ranged from 4 to 16 kb. We found that telomere shortening limits the replicative capacity of anthropoid fibroblasts and that the expression of human telomerase produced telomere elongation and the extension of their in vitro life span. In contrast the prosimian Lemur catta (ring-tailed lemur) had both long and short telomeres and telomere shortening did not provide an absolute barrier to immortalization. Following a transient growth arrest a subset of cells showing a reduced number of chromosomes overgrew the cultures without activation of telomerase. Here we show that the presence of continuous TTAGGG repeats at telomeres and rigorous control of replicative aging by telomere shortening appear to be conserved among anthropoid primates but is less effective in prosimian lemurs.     

Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding

Biotin carboxylases in mammalian cells are regulatory enzymes in lipogenesis and gluconeogenesis. In this study, endogenous biotin in skeletal and cardiac muscle was detected using avidin conjugated with alkaline phosphatase and applied in high concentrations to muscle sections. The avidin binding was subsequently visualized by histochemical demonstration of the alkaline phosphatase activity. All cardiac muscle cells showed high affinity for avidin with only the nuclei and the intercalated discs remaining unstained. In skeletal muscle a diffuse reaction could be detected in the sarcoplasm of the muscle fibres. A granular reaction was noted in the same fibres that showed activity for succinic dehydrogenase. The specificity of the coloured reaction product in the muscle sections was investigated and is suggested to be caused by avidin binding to biotin moieties in mitochondria and the cytosol. Mitochondrial and cytosolic preparations of skeletal muscle were electrophoresed in sodium dodecyl sulphate gels. After blotting and incubation with conjugated avidin, two bands with molecular weights of 75 kDa and 130 kDa respectively were evident in the mitochondrial preparation. It is suggested that the 75-kDa band represents comigration of the biotin-containing subunits of propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The 130-kDa band may represent the biotin-containing pyruvate carboxylase. In the cytosolic preparation a 270-kDa band was stained in blots that had been incubated with conjugated avidin; this band is suggested to represent acetyl-CoA carboxylase. A 190-kDa cytosolic band might be a cleavage product of acetyl-CoA carboxylase. We propose that using alkaline phosphatase-conjugated avidin it is possible to detect the mitochondrial and cytosolic biotin-dependent carboxylases in striated muscle.     

Discovery and evaluation of spirocyclic derivatives as antagonists of the neuropeptide Y5 receptor

A novel series of spirocyclic derivatives was synthesized and evaluated as NPY Y5R antagonists for the treatment of obesity. Cis and trans analogs 7a and 8a were equipotent in a Y5R binding assay (K(i)'s ≤ 1 nM) and displayed good stability in human and rat liver microsome preparations. Compound 7a failed to demonstrate weight loss activity in a diet-induced obese (DIO) rat model at unbound drug levels in the brain that exceeded the Y5R K(i) value by 25-fold over a 24-h time-period.     

An affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type 1 gp41 recognizes an epitope between those of 2F5 and 4E10

The membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 bears the epitopes of two broadly neutralizing antibodies (Abs), 2F5 and 4E10, making it a target for vaccine design. A third Ab, Fab Z13, had previously been mapped to an epitope that overlaps those of 2F5 and 4E10 but only weakly neutralizes a limited set of primary isolates. Here, libraries of Fab Z13 variants displayed on phage were engineered and affinity selected against an MPER peptide and recombinant gp41. A high-affinity variant, designated Z13e1, was isolated and found to be approximately 100-fold improved over the parental Fab not only in binding affinity for the MPER antigens but also in neutralization potency against sensitive HIV-1. Alanine scanning of MPER residues 664 to 680 revealed that N671 and D674 are crucial for peptide recognition as well as for the neutralization of HIV-1 by Z13e1. Ab competition studies and truncation of MPER peptides indicate that Z13e1 binds with high affinity to an epitope between and overlapping with those of 2F5 and 4E10, with the minimal peptide epitope WASLWNWFDITN. Still, Z13e1 remained about an order of magnitude less potent than 4E10 against several isolates of pseudotyped HIV-1. The sum of our molecular analyses with Z13e1 suggests that the segment on the MPER of gp41 between the 2F5 and 4E10 epitopes is exposed on the functional envelope trimer but that access to the specific Z13e1 epitope within this segment is limited. Thus, the ability of MPER-bearing immunogens to elicit potent HIV-1-neutralizing Abs may depend in part on recapitulating the particular constraints that the functional envelope trimer imposes on the segment of the MPER to which Z13e1 binds.     

Early activation of NK cells after lung infection with the intracellular bacterium, Francisella tularensis LVS

Francisella tularensis is a gram-negative intracellular bacterium that has been classified as a Category A biothreat because of its ability to induce deadly pneumonic tularemia when inhaled. In the present study, an experimental model of F. tularensis LVS intranasal infection was used to study the immune cells involved in cytokine secretion in the lungs after infection. Dramatic increases in the numbers of cells secreting IFN-gamma were observed 72 h after intranasal infection of BALB/c and C57BL/6 mice with sublethal (1000 CFU) or lethal (10,000 CFU) doses of F. tularensis LVS and the cells primarily responsible for this IFN-gamma expression were identified as CD11b+ DX5+ NK cells. The findings were further confirmed in C57BL/6 mice showing that cells responsible for IFN-gamma secretion in the lungs were CD11b+ DX5+ NK1.1+. NK cell depletion studies showed a decrease in the percentage of IFN-gamma secreting cells, due not only to a diminished proportion of IFN-gamma secreting NK cells, but also to a reduced percentage of T cells secreting IFN-gamma. The results indicate that IFN-gamma is secreted in response to respiratory infection with F. tularensis LVS, and that NK cells are the early responders responsible for IFN-gamma secretion.