Inhibition of Listeria monocytogenes in Cooked Ham by Virulent Bacteriophages and Protective Cultures

Protective cultures can be used successfully as an additional hurdle together with phages to reduce growth of Listeria monocytogenes on sliced cooked ham. Addition of phages resulted in a rapid 10-fold reduction of L. monocytogenes. After 14 to 28 days of storage, a 100-fold reduction was observed in samples with phages and protective culture compared to results for samples with phages alone.Listeriosis in Europe has an average incidence between 2 and 10 reported cases per million population per year (7). Listeria monocytogenes is found in raw and ready-to-eat (RTE) products, poultry, seafood, and dairy products. A review of the incidence and transmission of L. monocytogenes in RTE products has been published by Lianou and Sofos (11). The USDA has implemented a “zero-tolerance” policy for L. monocytogenes in RTE products (2). In the European Union, the limit for common RTE foods is 100 CFU/g (1). Recently, Codex Alimentarius adopted new standards for L. monocytogenes in RTE foods, with a limit of 100 CFU/g in foods where L. monocytogenes cannot grow and absence in foods where the bacterium can grow. However, an alternative approach is accepted. Competent authorities may choose to establish and implement other validated limits (http://www.codexalimentarius.net/web/archives.jsp?lang=en, Alinorm 09/32/REP and Alinorm 09/32/13). L. monocytogenes in cooked products is connected with cross-contamination after heat treatment (11, 12). Bacteriophages have been successfully applied to a number of food products to reduce the level of contaminating L. monocytogenes (6, 8-10, 13, 14). The effect of phages varies with the type of product and is strongly dose dependent (6, 8). Active phages can be recovered from foods after long storage, but the phage particles appear to become immobilized soon after addition to nonliquid foods and therefore, due to limited diffusion, cannot infect bacteria (8). Bacteria surviving phage treatment can later grow in the product. Additional hurdles should therefore be present to inhibit later outgrowth of L. monocytogenes.We have previously employed Lactobacillus sakei TH1 as a protective culture against L. monocytogenes (4, 5). Here we examine the combined use of phages and protective culture to reduce outgrowth of L. monocytogenes on cooked ham.Rifampin (rifampicin)-resistant mutants of L. monocytogenes 2230/92 serotype 1, implicated in a listeriosis outbreak in Norway (12), and L. monocytogenes 167 serotype 4b were grown overnight in brain heart infusion (Difco Laboratories, Detroit, MI) at 37°C without shaking and stored at 4°C for 24 h (3-5). Cells were diluted in 0.9% NaCl and plated on brain heart infusion agar with 200 μg/ml rifampin. L. sakei TH1 was grown at 30°C in MRS (de Man, Rogosa, Sharpe) broth (CM 359; Oxoid, Hampshire, England) (pH 6.2) and plated on MRS agar (5). Listex P100 phages, 2 × 10¹¹ PFU/ml, were from EBI Food Safety (Wageningen, The Netherlands).Ten-gram slices of hams with 2.3% NaCl and 0.01% disodium diphosphate (pH 6.2; a_w > 0.97), made at Nofima''s pilot plant (Aas, Norway), were inoculated with a cold-adapted 1:1 mixture of L. monocytogenes 2230/92 and 167. Bacteria were spread in 100 μl 0.9% NaCl over the 80-cm² surface area of each slice to 10³ CFU/cm² using a bent glass rod. After 1 h at 20°C, phages (5 × 10⁷ PFU/cm² in a total volume of 100 μl) were spread over the same surface. After one additional hour, 10³ CFU/cm² L. sakei TH1 in 100 μl 0.9% NaCl was added where appropriate. The slices were vacuum packed and stored at 10°C. Growth was measured before and after spiking and at 0, 3, 7, 14, and 28 days after homogenizing the slices in 100 ml 0.9% NaCl in a Stomacher homogenizer. No lactic acid bacteria were detected in uninoculated samples. Experiments were performed with three parallel samples. L. monocytogenes alone grew from 10⁴ CFU/g at the onset of the experiment to 10⁷ CFU/g the first 7 days, reached 2 × 10⁸ CFU/g after 14 days, and remained unchanged thereafter (Fig. ​(Fig.1).1). In samples with both L. monocytogenes and phages, a rapid 1-log reduction in L. monocytogenes was observed. Surviving L. monocytogenes, however, grew as well as that in the phage-free controls, reaching >10⁷ CFU/g after 14 days. In samples where both P100 phages and L. sakei TH1 were added, the same initial reduction of L. monocytogenes was observed, but the later outgrowth was reduced by the fast-growing lactic acid bacteria and the L. monocytogenes levels were 2 logs lower than those with P100 phages alone after 28 days of incubation. The phages did not influence the growth and survival of L. sakei TH1. During the 28 days of storage, the pH changed from 6.20 to 6.05 in samples with L. monocytogenes and to 6.00 in samples with both L. monocytogenes and L. sakei TH1. The results were reproduced in a separate repetition of the experiment at 10°C (not shown).Open in a separate windowFIG. 1.Inhibition of L. monocytogenes in cooked ham with bacteriophages and protective culture at 10°C. Sliced ham was inoculated with 10³ CFU/cm² (corresponding to approximately 10⁴ CFU/g) L. monocytogenes (⧫), L. monocytogenes and 5 × 10⁷ PFU/cm² P100 phages (▪), or L. monocytogenes, 5 × 10⁷ PFU/cm² P100 phages, and 10³ CFU/cm² (approximately 10⁴ CFU/g) protective-culture L. sakei TH1 (▴) and stored at 10°C. Growth of L. sakei TH1 is shown by the broken line.The effect of the protective culture was dose dependent when 10⁴ CFU/g and 10⁶ CFU/g of L. sakei TH1 were added to slices of ham (Fig. ​(Fig.2).2). L. monocytogenes alone grew to 2 × 10⁸ CFU/g after 14 days. When L. sakei TH1 was added at a low concentration (10⁴ CFU/g), L. monocytogenes grew to approximately 4 × 10⁶ CFU/g, while when L. sakei TH1 was added at a high concentration, L. monocytogenes levels were 1 to 2 logs lower. The pHs in the low- and high-inoculum hams were reduced from the initial 6.20 to 6.16 and 6.02, respectively, at day 28. For hams stored at 4°C, slow growth of L. monocytogenes occurred between days 14 and 28 from 10⁴ to 10⁵ CFU/g (P = 0.003) (Fig. ​(Fig.3).3). With phages and L. sakei TH1 added, a rapid 1-log reduction of L. monocytogenes was observed due to the phage attack, and no growth was observed during the 28-day storage period. The L. sakei TH1 strain showed a longer lag phase at this low temperature but nevertheless reached 10⁷ CFU/g at day 14 and thereby inhibited any growth of L. monocytogenes.Open in a separate windowFIG. 2.Inhibition of L. monocytogenes in cooked ham inoculated with large or small amounts of protective culture at 10°C. Sliced ham was inoculated with 10³ CFU/cm² (corresponding to approximately 10⁴ CFU/g) L. monocytogenes (⧫), L. monocytogenes and 10⁶ CFU/g (10⁵ CFU/cm²) L. sakei TH1 (▴), or L. monocytogenes and 10⁴ CFU/g (10³ CFU/cm²) L. sakei TH1 (▪) and stored at 10°C. Growth of L. sakei TH1 is shown by broken lines. The L. monocytogenes control is the same control as in Fig. ​Fig.11.Open in a separate windowFIG. 3.Inhibition of L. monocytogenes in cooked ham with bacteriophages and protective culture at 4°C. Sliced ham was inoculated with 10³ CFU/cm² (corresponding to approximately 10⁴ CFU/g) L. monocytogenes (⧫) or L. monocytogenes, 5 × 10⁷ PFU/cm² P100 phages, and 10⁴ CFU/g (10³ CFU/cm²) protective-culture, L. sakei TH1 (▴) and stored at 4°C. Growth of L. sakei TH1 is shown by the broken line.Since L. sakei TH1 grows well at low temperatures, prevents growth of L. monocytogenes, and has no negative influence on the organoleptic properties of ham (4, 5), it can successfully be employed as an additional hurdle together with phages.We here chose to perform the storage experiments under “worst-case” conditions. Generally, the contamination levels of L. monocytogenes are lower than in our setup, in the range of 10 to 100 CFU/g (see reference 11 and references therein). Since L. sakei TH1 grows well at low temperatures (Fig. ​(Fig.3),3), its selective advantage will be greater at 4°C than at abuse temperatures. From the above, it is evident that it is possible to optimize L. monocytogenes inhibition by increasing both the phage titer and the starting amount of protective culture. An enhanced effect may also be experienced by modifying phage application, e.g., by using larger liquid volumes (6, 8).Emergence of resistant L. monocytogenes may be a potential problem when treating foods with phages. No emergence of resistance has been detected after phage treatment (6, 8). Such strategies as use of phage mixtures, phage rotation schemes, and treatment of products immediately prior to packaging may reduce eventual resistance problems (8). Some L. monocytogenes strains are naturally phage resistant (6). In these cases, a protective culture still constitutes a powerful hurdle.In conclusion, we have shown here that by applying phages and protective culture as two independent hurdles, it is possible to both reduce the number of L. monocytogenes bacteria on a product and inhibit outgrowth of eventual remaining surviving cells. This is a general method that can potentially be applied to different foods where there is a potential risk for growth of L. monocytogenes, provided a suitable protective culture is available.     

Identification of Carnobacterium spp. and Leuconostoc spp. in meat by genus-specific 16S rRNA probes

Oligonucleotide probes specific for Carnobacterium and Leuconostoc species were constructed from the variable regions of 16S rRNA obtained from the literature and sequence data bases. The probes were hybridized with crude nucleic acid extract from 32 type strains of lactic acid bacteria (LAB) commonly found on meat. Two of the probes hybridized only to the four Carnobacterium species whereas the other two hybridized only to five of the six Leuconostoc species tested. The probes were also hybridized with nucleic acids from unknown strains of LAB. The identification was consistent with the results of biochemical tests used to characterize the two genera.     

Cloning, sequencing and expression of the gene encoding the cell-envelope-associated proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151.

The gene encoding the cell-envelope-associated proteinase of Lactobacillus paracasei subsp. paracasei NCDO 151 (formerly Lactobacillus casei NCDO 151) was cloned and sequenced. The gene was located on the chromosome and encoded a polypeptide of 1902 amino acids. The proteinase is N-terminally cleaved upon maturation. It shows extensive homology to the Lactococcus lactis subsp. cremoris Wg2 proteinase. Similar to the situation in Lactococcus, a maturation gene was found upstream of the proteinase gene. The cloned proteinase gene was expressed in Lactobacillus plantarum. However, no expression was observed when the gene was cloned in Lactococcus lactis.     

Lipogenesis in liver and adipose tissue of the domestic cat (Felis domestica)

1. Production of fatty acids, CO2 and glycerol from glucose and acetate was determined in slices of liver and adipose tissue taken from mature cats. 2. Acetate was the predominant carbon source for de novo fatty acid synthesis in both liver and adipose tissue. 3. Fatty acid synthesis occurred at much greater rates in adipose tissue than in liver. 4. Relative lipogenic capacity (ratio of production of long-chain fatty acids and glycerol to oxidation of substrates) was 20 times greater in adipose tissue than in liver with both substrates.