Shelf life and safety aspects of chilled cooked and peeled shrimps (Pandalus borealis) in modified atmosphere packaging

AIMS: To evaluate the growth of Listeria monocytogenes and shelf life of cooked and peeled shrimps in modified atmosphere packaging (MAP). METHODS AND RESULTS: Storage trials with naturally contaminated cooked and peeled MAP shrimps (Pandalus borealis) were carried out at 2, 5 and 8 degrees C. Challenge tests at the same conditions were performed after inoculation with Listeria monocytogenes. Both storage trials and challenge tests were repeated after 4 months of frozen storage (-22 degrees C). Brochothrix thermosphacta and Carnobacterium maltaromaticum were responsible for sensory spoilage of cooked and peeled MAP shrimps. In challenge tests, growth of L. monocytogenes was observed at all of the storage temperatures studied. At 5 and 8 degrees C the concentration of L. monocytogenes increased more than a 1000-fold before the product became sensory spoiled whereas this was not observed at 2 degrees C. Frozen storage had only a minor inhibiting effect on growth of L. monocytogenes in the thawed product. CONCLUSIONS: To prevent L. monocytogenes becoming a safety problem, cooked and peeled MAP shrimps should be distributed at 2 degrees C and with a maximum shelf life of 20-21 d. At higher temperatures shelf life is significantly reduced. SIGNIFICANCE AND IMPACT OF THE STUDY: Information is provided to establish shelf life of cooked and peeled MAP shrimps.     

Fate of Listeria spp. on parsley leaves grown in laboratory and field cultures

AIMS: To investigate the population dynamics of Listeria monocytogenes and Listeria innocua on the aerial surfaces of parsley. METHODS AND RESULTS: Under 100% relative humidity (RH) in laboratory and regardless of the inoculum tested (10(3)-10(8) CFU per leaf), counts of L. monocytogenes EGDe, LO28, LmP60 and L. innocua CIP 80-12 tended towards approx. 10(5) CFU per leaf. Under low RH, Listeria spp. populations declined regardless to the inoculum size (10(4)-10(8) CFU per leaf). L. innocua CIP 80-12 survived slightly better than L. monocytogenes in the laboratory and was used in field cultures. Under field cultures, counts of L. innocua decreased more rapidly than in the laboratory, representing a decrease of 9 log(10) in 2 days in field conditions compared to a decrease of 4.5 log(10) in 8 days in the laboratory. Counts of L. innocua on tunnel parsley cultures were always higher (at least by 100 times) than those on unprotected parsley culture. CONCLUSIONS: Even with a high inoculum and under protected conditions (i.e. plastic tunnels), population of L. monocytogenes on the surface of parsley on the field would decrease by several log(10) scales within 2 days. SIGNIFICANCE AND IMPACT OF THE STUDY: Direct contamination of aerial surfaces of parsley with L. monocytogenes (i.e. through contaminated irrigation water) will not lead to contaminated produce unless it occurs very shortly before harvest.     

Limitations in the use of Drosophila melanogaster as a model host for gram-positive bacterial infection

AIMS: To examine sensitivities of various Drosophila melanogaster strains towards human pathogenic and nonpathogenic gram-positive bacteria. METHODS AND RESULTS: The D. melanogaster Oregon R strain was infected by injecting the thorax with a needle containing Escherichia coli (negative control), Listeria monocytogenes, Staphylococcus aureus (both food-borne pathogens), Listeria innocua, Bacillus subtilis, Carnobacterium maltaromaticum, Lactobacillus plantarum or Pediococcus acidilactici (all nonpathogenic bacteria). Listeria monocytogenes and S. aureus killed the host rapidly compared with the negative control. Infection with L. innocua, B. subtilis or C. maltaromaticum also resulted in a high fly mortality, whereas Lact. plantarum and P. acidilactici resulted in a slightly increased mortality. Four additional D. melanogaster lines, three of which had been selected for heat, cold and desiccation resistance respectively, were subjected to infection by L. monocytogenes, S. aureus and E. coli. Mortality rates were comparable with that of the Oregon R strain. CONCLUSIONS: Use of the injection method shows the limitation of D. melanogaster as a model host for gram-positive bacteria as opportunistic infection by nonpathogenic gram-positive bacteria results in partial or high mortality. In addition, lines of fruit flies resistant to various stress exposures did not show an increased resistance to infection by gram-positive pathogens under the conditions tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the inadequacy of D. melanogaster infected by the injection method in order to distinguish between virulent and nonvirulent gram-positive bacteria.     

Antimicrobial resistance of Listeria spp. recovered from processed bison

AIMS: The current study examined the antimicrobial susceptibility of 86 Listeria spp. isolated from processed bison carcasses. MATERIALS AND METhods: Susceptibility to 25 antimicrobial agents was determined using E-test and National Antimicrobial Resistance Monitoring System (NARMS) panels. Most Listeria isolates (88-98%) exhibited resistance to bacitracin, oxacillin, cefotaxime, and fosfomycin. Resistance to tetracycline (18.6%) was also common. Of the 16 tetracycline-resistant Listeria isolates, 15 carried tetM and 2 contained integrase of Tn1545 transposons. Rifampicin and trimethoprim-sulfamethoxazole were the most active antimicrobial agents against Listeria spp., with a MIC(90) of 0.38 microg ml(-1). Ampicillin, erythromycin, penicillin, gentamicin, and tobramycin also exhibited good activity against Listeria spp., with MIC(90) not exceeding 1 microg ml(-1). Differences in resistance among Listeria spp. was displayed, as Listeria innocua strains were more resistant than other Listeria species. CONCLUSIONS: The study showed that Listeria monocytogenes strains from bison were susceptible to the antibiotics most commonly used to treat human listeriosis. However, the presence of antimicrobial resistance in L. innocua indicates the potential for transfer of resistance and a conjugative transposon to L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of our study will provide useful information for the development of public health policy in the use of antimicrobials in food animal production.     

Comparison of selective and non-selective enrichment media in the detection of Listeria monocytogenes from meat containing Listeria innocua

AIMS: This study investigated whether the higher incidence of recovery from meat of Listeria innocua compared with L. monocytogenes could be due to the laboratory media used, leading to an artificially lower detection of the pathogenic species, L. monocytogenes. METHODS AND RESULTS: Minced beef was inoculated with L. monocytogenes, L. innocua, or a mixture of these species, and stored at 0 or 10 degrees C under vacuum or aerobic conditions for up to 28 days. Listeria were recovered from the minced beef using selective (University of Vermont Medium, UVM) and non-selective (Buffered Peptone Water, BPW) enrichment broths after 0, 14, and 28 days of storage. In general, there were no significant differences (P < 0.05) between the numbers of L. monocytogenes recovered from minced beef samples after 24 h enrichment in BPW and the numbers recovered using UVM. In addition, the presence of L. innocua in meat samples containing L. monocytogenes did not significantly (P < 0.05) affect the numbers of L. monocytogenes recovered using either enrichment broth. In most cases there were no significant differences (P < 0.05) between the numbers of L. innocua recovered from minced beef samples after 24 h enrichment in BPW compared with numbers recovered using UVM. CONCLUSION: Listeria innocua was found to have no significant competitive advantage over L. monocytogenes in selective or non-selective enrichment media. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that, in some instances, the use of a selective enrichment broth offers no advantage over a non-selective enrichment broth for the recovery of Listeria species from minced beef.     

Survival of Listeria innocua on hot and cold beef carcass surfaces

AIMS: This study aimed to determine the survival and growth of Listeria innocua on hot and cold beef carcass surfaces. METHODS AND RESULTS: Four sites, the neck, outside round, brisket and foreshank/brisket, were inoculated with L. innocua (i) immediately after dressing while hot and (ii) when cold after chilling. After inoculation, all carcasses were stored at 4 degrees C for 72 h. Survival of L. innocua on cold surfaces declined during storage and was less than on hot carcasses at all times. Data on the survival of L. innocua in broth (maximum recovery diluent) indicated that counts could not be compared with those on carcasses, in particular on cold carcasses. CONCLUSIONS: The results indicate that L. innocua survives on hot carcass surfaces during chilling, but declines over time on cold surfaces. The decrease in L. innocua counts on cold surfaces may be related to a synergy between the combined stresses of low available water (a(w)) and low temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to determine the effect of chilling on the survival and growth of Listeria on beef carcass surfaces. The information can potentially be used to determine the survival and growth of the pathogen, L. monocytogenes on beef surfaces.     

Evaluation of the API test, phosphatidylinositol-specific phospholipase C activity and PCR method in identification of Listeria monocytogenes in meat foods

The aim of this work was to compare the possibility of identifying Listeria monocytogenes strains isolated from meat and sausage on the basis of the API-Listeria test, production of phosphatidylinositol-specific phospholipase C (PI-PLC) and polymerase chain reaction (PCR) for a DNA fragment of the hlyA gene encoding listeriolysin O. Forty-six strains were isolated and examined. The lethality of some Listeria isolates for BALB/c mice was also determined. In this study, all isolates identified as L. monocytogenes in the API test gave a positive signal in the PCR. Listeriae identified as L. innocua or L. welshimeri in the API test were negative in the PCR conducted with the primers for listeriolysin O. All strains identified as L. monocytogenes on the basis of the API test and the PCR produced PI-PLC. However, this activity was not limited to the bacteria of this species. Four out of 17 L. innocua and three out of 10 L. welshimeri isolates were PI-PLC-positive. None of the L. innocua or L. welshimeri isolates (neither PI-PLC+ or PI-PLC-) showed lethality for BALB/c mice. In contrast, two L. monocytogenes isolates as well as a reference L. monocytogenes strain killed all mice used for the experiment.     

Behaviour of the pathogen surrogates Listeria innocua and Clostridium sporogenes during production of parsley in fields fertilized with contaminated amendments

The survival and transfer of Listeria innocua and Clostridium sporogenes, used as surrogates of the food borne pathogens Listeria monocytogenes and Clostridium botulinum, were quantitatively assessed under field conditions. In the soil, spores of C. sporogenes declined by less than 0.7 log cycles within 16 months and were detected on parsley leaves throughout the experiment. In contrast, L. innocua in the soil declined by 7 log cycles in 90 days and was detected on leaves in low numbers (>0.04 MPN g(-1)) during the first 30 days. Rates of decline in soil were similar in the laboratory at 20 degrees C for two strains of L. innocua and L. monocytogenes ; and in the field for L. innocua over two different years. L. innocua survived better in winter, indicating an important influence of temperature. The major cause of transfer of L. innocua from soil to parsley leaves was splashing due to rain and irrigation. As few as 1 CFU g(-1) Listeria in soil led to contamination of parsley leaves. Internalisation of Listeria through parsley roots was not observed. Under the conditions of soil and climate studied, a delay of 90 days between application of potentially contaminated fertilizer and harvest should be sufficient to eliminate L. monocytogenes.     

Effects of iron and oxygen species scavengers on Listeria spp. chemiluminescence

Listeria monocytogenes and Listeria innocua are able, under certain conditions, to produce chemiluminescence (CL), which is amplified by luminol. Kinetic studies of CL by L. monocytogenes and L. innocua show a close parallelism between CL and growth curves during the exponential phase, with a maximum of CL reached just before entrance of bacteria into the stationary phase. CL is tightly correlated with the release of oxygen compounds. The reactive oxygen species scavengers tryptophan, mannitol, and tiron, as well as cellobiose and high temperature, were assessed with regard to CL in the two Listeria species. Only tiron strongly reduced the CL emitted by L. monocytogenes and L. innocua. On the other hand, charcoal pretreatment of the growth medium inhibited the CL, whereas ferric citrate strongly increased the CL of L. monocytogenes and L. innocua. These data suggest that iron and superoxide radical are implicated in the CL produced by these bacteria, but this phenomenon is not correlated to virulence.     

李斯特菌毒力因子及其进化

李斯特菌属包含6个种,毒力各有差异。在细菌耐受外界环境、黏附侵袭及细胞内感染过程中,毒力因子各司其职又相互协作。毒力基因常聚集为毒力岛,其中PrfA依赖型毒力基因簇(LIPI-1)与内化素岛(LIPI-2)是致病种最重要的两个毒力岛。李斯特菌各个种可能来源于同一个携带有完整毒力岛的祖先,在长期进化过程中,通过基因水平转移或重组、整合等事件,演化为目前流行的6个种。噬菌体、转座子、质粒等可能扮演着毒力进化执行者的角色。一些天然非典型菌株是目前研究的热点,如含有LIPI-1的无害李斯特菌和缺失LIPI-1的塞氏李斯特菌,其演化进程可能尚未达到或已超越目前流行的状态,为李斯特菌毒力进化的研究提供了重要线索。     

Single cell variability of L. monocytogenes grown on liver pâté and cooked ham at 7 degrees C: comparing challenge test data to predictive simulations

AIMS: The variability in growth between individual Listeria monocytogenes cells was investigated on liver paté and cooked ham. These results were compared to Monte Carlo simulations based on data collected previously in broths (Francois et al., submitted for publication). METHODS AND RESULTS: Single cells were isolated by a dilution protocol and inoculated on 15 g samples of liver paté and cooked ham, pasteurized in the packaging. Of each product, 250 samples were inoculated, of which 50 samples were analysed for L. monocytogenes on each analysis day. Results were compared to simulations, based on distributions that describe the variability of the individual cell lag phases and generation times of L. monocytogenes cultivated in broths. Based on the same simulation techniques, the variability effect was investigated for different inoculum levels (10, 100, 10,00 and 10,000 cells). It was demonstrated that the expected variability of the outgrowth of L. monocytogenes in a challenge test is very high for low inoculum levels. CONCLUSIONS: The variability in growth characteristics observed between different single L. monocytogenes cells on foods is very large. The simulations based on the previously collected optical density data in broths, could be confirmed by foods inoculated with single L. monocytogenes cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The large variability between different individual L. monocytogenes cells has serious consequences for the experimental design of a challenge test. One thousand cells per portion are necessary in order to reduce the variability to acceptable levels and quantify the behaviour of the pathogen consistently with a reasonable number of challenge tests.     

Antibiotic resistance among Listeria, including Listeria monocytogenes, in retail foods

AIMS: In the past eight to 10 years, reports of antibiotic resistance in food-borne isolates in many countries have increased, and this work examined the susceptibility of 1001 food isolates of Listeria species. METHODS AND RESULTS: Susceptibility/resistance to eight antibiotics was determined using the Bauer-Kirby disc diffusion assay, and 10.9% of the isolates examined displayed resistance to one or more antibiotics. Resistance to one or more antibiotics was exhibited in 0.6% of Listeria monocytogenes isolates compared with 19.5% of Listeria innocua isolates. Resistance was not observed in Listeria seeligeri or Listeria welshimeri. Resistance to tetracycline (6.7%) and penicillin (3.7%) was the most frequently observed, and while resistance to one antibiotic was most common (9.1%), isolates resistant to two or more antibiotics (1.8%) were also observed. CONCLUSION: While resistance to the antibiotics most commonly used to treat human listeriosis was not observed in L. monocytogenes, the presence of such resistance in other Listeria species raises the possibility of future acquisition of resistance by L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The higher level of resistance in L. innocua compared with L. monocytogenes suggests that a species-related ability to acquire resistance to antibiotics exists.     

Evaluation of ALOA plating medium for its suitability to recover high pressure-injured Listeria monocytogenes from ground chicken meat

AIMS: To evaluate a chromogenic plating medium for the isolation of sublethally injured cells of Listeria monocytogenes from processed foods. METHODS AND RESULTS: The inactivation of L. monocytogenes at pressures up to 400 MPa and 12 degrees C in ground chicken meat was employed to examine the recovery of high-pressure injured cells. Before and after different repair incubation periods at 30 degrees C in a nonselective broth, samples were plated onto a selective and differential agar [Agar Listeria according to Ottaviani and Agosti (ALOA)] and in the same medium supplemented with 4% sodium chloride (ALOA-S), and incubated at 37 degrees C. Sublethally injured cells were able to grow when directly plated onto the ALOA medium, without a previous repair incubation period. However, only uninjured cells grew on the ALOA-S medium. CONCLUSIONS: Sublethally injured cells of L. monocytogenes can be quantified by subtracting counts on ALOA-S medium from counts on ALOA medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Possible applications include direct enumeration on ALOA of stressed cells of L. monocytogenes in foods with more than 100 colony forming units per gram.     

Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: clues for evolution and pathogenicity

Listeria monocytogenes, an invasive opportunistic, food-borne pathogen, remains one of the leading causes of mortality from food-borne infections. The recently determined complete genome sequences of L. monocytogenes strain EGDe and of that of the closely related non-pathogenic species Listeria innocua strain CLIP11262 enhance our knowledge of the genetic basis of the virulence of L. monocytogenes and advance our understanding of the evolution of these Listeria species. Both genomes encode a high number of surface, transport and regulatory proteins. Comparison of the genome organisation revealed a perfect synteny between the two Listeria genomes. Comparison with other closely related bacteria also showed a high conservation in genome organisation among the Listeria, Staphylococcus and Bacillus group of low G+C content bacteria. Distinct G+C content of a number of strain-specific genes suggests intensive lateral gene transfer. The identification of a 55-kb locus encoding proteins with high homology to Salmonella enterica serovar Typhimurium vitamin B(12) synthesis proteins as well as those necessary for degradation of ethanolamine and propanediol further indicates acquisition of a complete metabolic pathway by horizontal gene transfer and a probable role of this locus in anaerobic growth in the host.     

Natural atypical Listeria innocua strains with Listeria monocytogenes pathogenicity island 1 genes

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentation-negative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.     

Heteroduplex mobility assay for the identification of Listeria sp and Listeria monocytogenes strains: application to characterisation of strains from sludge and food samples

One hundred and ten Listeria sp. isolates from sewage sludge were identified according to phenotypic and genotypic methods. The Listeria sp. strains isolated from five types of sludge from three sewage treatment plants in Angers (France) and the surrounding area included L. monocytogenes (55.5%), L. innocua (29.1%), L. seeligeri (13.6%) and L. welshimeri (1.8%). The majority of L. monocytogenes strains belonged to serotypes 4b, 1/2b and 1/2a. Moreover, a heteroduplex mobility assay based on the 16S rRNA sequences was tested for its ability to identify the six species of the genus Listeria. This study, performed on 283 Listeria sp. strains from human, food and sewage sludge samples, showed that all the species were distinguishable from one another. L. innocua and L. seeligeri showed respectively three and two distinct banding patterns. Within L. monocytogenes, four groups (I-IV) were defined. The majority of food and environmental isolates were clustered in group I and it is noteworthy that group IV clustered epidemiologic isolates and strains belonging to serotypes 4b, 1/2a and 1/2b.     

The incidence of Listeria spp. in soft cheeses, butter and raw milk in the province of Bologna

Samples of soft cheese, butter and raw milk were examined for Listeria species. Listeria monocytogenes (serotype 1, haemolytic and virulent for mice) and L. innocua (the only other Listeria sp. isolated) were each found in 2/21 (1.6%) of soft cheese samples. Five per cent of butter samples were contaminated with L. innocua. No Listeria spp. were detected in 40 raw milk samples. The results were compared with similar studies in Italy and abroad.     

An improved selective isolation medium for the recovery of Listeria monocytogenes from smoked fish

AIMS: The aim of this study was to improve the selective isolation of Listeria monocytogenes from smoked haddock fillets. METHODS AND RESULTS: Listeria selective agar (LSA)--Oxford formulation was supplemented with 25 microg x ml(-1) of colistin sulphate and 30 microg x ml(-1) of nalidixic acid. Inocula from four smoked haddock fillets produced colonies (approx. 2-13 bacteria x g(-1)), identified as L. monocytogenes, on LSA supplemented with antimicrobial compounds (MLSA). Moreover, there was only negligible evidence of bacteria which were not L. monocytogenes on MLSA. In contrast, LSA supported dense bacterial growth, which was not equated with L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The modified medium permitted the recovery of L. monocytogenes from smoked haddock fillets and reduced the growth of contaminating bacteria.     

The incidence of Listeria spp. in soft cheeses, butter and raw milk in the province of Bologna

Samples of soft cheese, butter and raw milk were examined for Listeria species. Listeria monocytogenes (serotype 1, haemolytic and virulent for mice) and L. innocua (the only other Listeria sp. isolated) were each found in 2/21 (1.6%) of soft cheese samples. Five per cent of butter samples were contaminated with L. innocua . No Listeria spp. were detected in 40 raw milk samples. The results were compared with similar studies in Italy and abroad. and accepted 14 June 1989