title: Evidence of metabolically active but non-culturable Listeria monocytogenes in long-term growth at 10 °C.
abstract: Cultures of Listeria monocytogenes at low temperatures (10 °C) in a broth model revealed long-term survival at about 0.04% cell density in relation to the log phase. In contrast, direct viable counts and PMA real-time PCR data suggested that 50% and 1% of the population retain membrane integrity, respectively. To elucidate the observed difference, the metabolic activity of the bacterial population was investigated by multiparametric flow cytometry, including the assessment of membrane integrity, reductase activity, as well as forward and side scatter properties. These analyses were complemented by 16S rRNA real-time PCR. The majority of the cells retained their membrane integrity and reductase activity until day 29. On day 42, 48.00 ± 4.00% (L. monocytogenes strain 3251) and 68.67 ± 3.74% (L. monocytogenes strain 535) of the cells had intact membranes, whereas 57.23 ± 1.85% (strain 3251) and 74.97 ± 3.01% (strain 535) exhibited high reductase activity. On day 42, mean 16S rRNA copy numbers of 3.98 ± 1.37 (membrane integrity) and 3.86 ± 1.32 (reductase activity) remained per intact or active cell. Our data suggest the transition of L. monocytogenes into a state of metabolic dormancy during long-term culture at low temperature.
authors.affiliation: Institute of Milk Hygiene, Milk Technology and Food Safety, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria. Electronic address: anna.gurresch@hotmail.com.
authors.forename: Anna
authors.initials: A
authors.lastname: Gurresch
authors.affiliation: Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria. Electronic address: wilhelm.gerner@vetmeduni.ac.at.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Gerner
authors.affiliation: Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom. Electronic address: carmen.pin@ifr.ac.uk.
authors.forename: Carmen
authors.initials: C
authors.lastname: Pin
authors.affiliation: Institute of Milk Hygiene, Milk Technology and Food Safety, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria. Electronic address: martin.wagner@vetmeduni.ac.at.
authors.forename: Martin
authors.initials: M
authors.lastname: Wagner
authors.affiliation: Institute of Milk Hygiene, Milk Technology and Food Safety, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria. Electronic address: ingeborg.hein@gmx.at.
authors.forename: Ingeborg
authors.initials: I
authors.lastname: Hein
mesh_terms: D058491:Bacterial Load; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology; D003080:Cold Temperature; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005434:Flow Cytometry; D008089:Listeria monocytogenes / Q000254:growth & development* / Q000502:physiology* / Q000528:radiation effects; D050296:Microbial Viability / Q000528:radiation effects*; D010088:Oxidoreductases / Q000032:analysis; D010539:Permeability; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D010088:Oxidoreductases
keywords: Decline; Fluorescence cytometry; Listeria monocytogenes; Long-term survival; Metabolic activity