Lactic acid bacteria from chicken carcasses with inhibitory activity against Salmonella spp. and Listeria monocytogenes

This study was conducted to isolate psychrotrophic lactic acid bacteria (LAB) from chicken carcasses with inhibitory activity against strains of Salmonella spp. and Listeria monocytogenes. A total of 100 broiler samples were examined for the presence of LAB. Ninety-two LAB isolates that showed antimicrobial effects against Salmonella spp. and L. monocytogenes were further analysed to examine their LAB (Gram-positive, catalase negative, oxidase negative) and psychrotrophic characteristics (ability to grow at 7 °C). Fifty isolates were further selected and identified initially using standard biochemical tests in miniature (Micro-kits API CH 50) and then by sequencing of the 16s-23s rRNA gene boundary region (Intergenic Spacer Region). By molecular identification, these isolates were classified into 5 different LAB species: Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus johnsonii, Pediococcus acidilactici, and Lactobacillus paralimentarius. None of the isolates produced tyramine or histamine.     

Application of antimicrobial-producing lactic acid bacteria to control pathogens in ready-to-use vegetables

Five psychrotrophic strains of lactic acid bacteria (Lactobacillus casei, Lact. plantarum and Pediococcus spp.) were isolated from 22 samples of commercial salads. These strains were shown to inhibit Aeromonas hydrophila, Listeria monocytogenes, Salmonella typhimurium and Staphylococcus aureus on MRS agar, in salads and in juice prepared from vegetable salads. Lactobacillus casei IMPCLC34 was most effective in reducing total mesophilic bacteria and the coliform group; Aer. hydrophila, Salm. typhimurium and Staph. aureus disappeared after 6 d of storage, while the counts for L. monocytogenes remained constant. The potential application of antimicrobial-producing lactic acid bacteria as biopreservatives of ready-to-use vegetables is suggested.     

Bacteriocinogenic activity of lactobacilli from fermented sausages

During the screening of the inhibitory activity of 254 strains of lactobacilli isolated from fermented sausages at different times of ripening, 22% of the strains showed inhibition that was not related to acid or hydrogen peroxide, towards one or more indicator strains. Not all the strains were capable of secreting the inhibitory compound in the supernatant fluid. The characterization of the inhibitory compound from three strains showed that they were bacteriocins with a bactericidal mode of action and a molecular weight exceeding 10000 Da. Lactobacillus plantarum CTC 305, CTC 306 and Lact. sake CTC 372 inhibited Listeria monocytogenes. Lactobacillus sake CTC372 was cured of two plasmids of 84.8 kbp and 41.3 kbp, losing the production and the immunity of a bacteriocin as well as the ability to ferment lactose.     

Bacteriocin-like substance (BLS) production in Aeromonas hydrophila water isolates

30 Aeromonas hydrophila water isolates were tested for bacteriocin-like substance (BLS) production using a target panel of closely related microorganisms and other Gram-positive and Gram-negative bacteria, including food-borne pathogens. A. hydrophila showed antibacterial activity against one or more indicator microorganisms, but the activity emerged only with non-phylogenetically related genera or species. In particular all A. hydrophila showed antibacterial activity against one or more of the tested Staphylococcus strains, five against Listeria spp. (Listeria seeligeri, Listeria welshimeri and Listeria ivanovii), and eight presented a weak antagonistic activity towards Streptococcus agalactiae and Lactobacillus spp. Inhibitory activity was not observed against the other Gram-positive (Listeria monocytogenes, Listeria innocua and Enterococcus spp.) and Gram-negative tested strains, including Aeromonas sobria, Aeromonas caviae and the same A. hydrophila, when used as indicator. Anti-staphylococcal activity was observed with a gradual increase of the inhibition zone during incubation and seemed to be influenced by A. hydrophila hemolytic expression. Extrachromosomal analysis showed the presence, in 70% of the strains, of one to five plasmids with molecular masses ranging from 2.1 to 41.5 MDa, but it was not possible to relate this result with BLS production.     

Inhibition of Listeria in dry fermented sausages by the bacteriocinogenic Lactobacillus sake CTC494

Lactobacillus sake CTC494 isolated from a naturally fermented sausage, produced an antibacterial agent active against selected strains of Listeria monocytogenes and L. innocua. The agent was bacteriolytic against L. monocytogenes and sensitive to proteolytic enzymes; it was identified as a bacteriocin and was designated as sakacin K. The ability of Lact. sake CTC494 to inhibit the growth of listeria, compared to a bacteriocinogenic negative control strain, was examined in a model sausage system and in dry fermented sausages. In dry fermented sausages Lact. sake CTC494 was able not only to suppress the growth of listeria but to diminish their number by 1.25 log compared to the non-bacteriocinogenic control strain. Thus, Lact. sake CTC494 has proved to be a good starter culture providing good organoleptical and sensorial qualities to the product and can be employed as a bioprotective starter culture in fermented meat products.     

Development of a rapid method for the identification of Lactobacillus spp. isolated from naturally fermented italian sausages using a polymerase chain reaction-temperature gradient gel electrophoresis

A rapid method for the identification of Lactobacillus spp. isolated from naturally fermented Italian sausages was developed. It is based on the amplification of a small fragment from the 16S rRNA gene followed by temperature gradient gel electrophoresis (TGGE). Lactobacillus sakei, L. curvatus, L. alimentarius, L. casei, L. plantarum and L. brevis, obtained from International Collections, were used to optimize the method. Thiry-nine strains of Lactobacillus spp. were isolated from naturally fermented sausages and, after traditional identification, were tested by the PCR-TGGE protocol developed. No differences were observed comparing the results obtained, apart from five strains identified as L. curvatus that showed a PCR-TGGE profile identical to L. sakei.     

Fructooligosaccharides metabolism and effect on bacteriocin production in Lactobacillus strains isolated from ensiled corn and molasses

Fructo- (FOS) and galacto-oligosaccharides have been used to promote the growth of probiotics, mainly those from Lactobacillus genus. However, only few reports have evaluated the effect of prebiotics on bacteriocins activity and production. In this work, we characterized the effect of FOS supplementation on the growth, lactic and acetic acids production, and antimicrobial activity of crude extracts obtained from Lactobacillus strains isolated from ensiled corn and molasses. Seven out of 28 isolated Lactobacillus, belonging to Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus brevis, showed antimicrobial activity against Listeria innocua. Among them, the strain L. plantarum LE5 showed antimicrobial activity against Listeria monocytogenes and Enteroccocus faecalis; while the L. plantarum LE27 strain showed antimicrobial effect against L. monocytogenes, E. faecalis, Escherichia coli and Salmonella enteritidis. This antimicrobial activity in most of the cases was obtained only after FOS supplementation. In summary, these results show the feasibility to increase the antimicrobial activity of Lactobacillus bacteriocins by supplementing the growth medium with FOS.     

Distribution of pathogen inhibition in the Lactobacillus isolates of a commercial probiotic consortium

Pure strains of Lactobacillus ssp. isolated from a commercial probiotic consortium were checked in a double layer solid medium for their inhibition activities against selected pathogenic bacteria including serotypes of Listeria monocytogenes, Escherichia coli and Salmonella. The antagonistic properties of the Lactobacillus strains may be related to the production of bacteriocin-like compounds. All the pathogens tested were inhibited by one or a few strains of Lactobacillus, the best inhibition was observed against L. monocytogenes but the inhibition was also satisfactory against E. coli, Salm. typhimurium and Salm. enteritidis.     

The prevalence and number of Salmonella in sausages and their destruction by frying,grilling or barbecuing

AIMS: To determine the prevalence and number of Salmonella and Campylobacter in sausages and to evaluate their destruction during cooking. METHODS AND RESULTS: One hundred and sixty-two packs of uncooked economy or catering sausages, comprising 53 packs of frozen and 109 of chilled sausages, were purchased in Devon between March and July 2000. All were tested for the presence of Salmonella and 51 packs of chilled sausages were also examined for the presence of Campylobacter spp. To investigate the heat tolerance of Salmonella enterica serovar Typhimurium DT104 in sausage-meat, chilled, handmade and frozen sausages were inoculated with approx. 1.5 x 10(4) bacterial cells per sausage (approximately 300 cfu g(-1)) and then cooked by frying, grilling or barbecuing. The levels of creatinine kinase, lactate dehydrogenase and alkaline phosphatase in uncooked and cooked sausages were measured to evaluate their potential as indicators of adequate cooking and, therefore, pathogen elimination. Salmonella were detected in 7.5% of frozen and 9.1% of the chilled sausages (8.6% overall) but Campylobacter spp. were not isolated. After cooking, a visual assessment suggested that all of the sausages were thoroughly cooked. Despite this, barbecuing and frying sometimes allowed Salmonella cells to survive and the temperature profiles during cooking indicated that the lethal range was sometimes not reached. The enzyme levels tested were not reliable indicators of the inactivation of bacterial pathogens because Salmonella were sometimes isolated from sausages with low values of all three enzymes. CONCLUSIONS: Salmonella spp. are present in a significant proportion of sausages and are not always killed during the cooking process. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings have clear implications for public health.     

Isolation and identification of tetracycline resistant lactic acid bacteria from pre-packed sliced meat products

In recent years, the food chain has been recognised as one of the main routes for transmission of antibiotic resistant bacteria between the animal and human population. In this regard, the current study aimed to investigate if tetracycline resistant (tetR) lactic acid bacteria (LAB) are present in ready-to-eat modified atmosphere packed (MAP) sliced meat products including fermented dry sausage, cooked chicken breast meat and cooked ham. From population graphs based on doubling tetracycline concentrations between 0 and 256 microg ml(-1), only fermented dry sausage was shown to contain a high-level retR LAB population (5.10(1) - 2,23.10(4) CFU/g), and this in four out of ten examined sausages. From these four positive sausages, a total of 100 strains were isolated on de Man, Rogosa and Sharpe-sorbic acid (MRS-S) agar without tetracycline (n = 45) and on MRS-S agar supplemented with a tetracycline breakpoint concentration of 64 microg ml(-1) (n = 55). Using resistance histograms derived from the disc diffusion method, all these strains were grouped as sensitive to rifampicin, erythromycin and ampicillin. All strains from the tetracycline-containing MRS-S plates were resistant to tetracycline. Identification with whole-cell protein profiling revealed that the total strain set represented four different species: Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus sakei subsp. carnosus and Lactobacillus curvatus. All species are commonly associated with fermented dry sausage, either as starter culture or as natural contaminants. The latter three species were found to comprise all tetracycline resistant strains. To our knowledge, this is the first report providing evidence for the presence of tetR LAB in final ready-to-eat pre-packed fermented dry sausages.     

Antimicrobial potentiality of a phenothiazine group of antipsychotic drug-prochlorperazine

The antipsychotic drug, prochlorperazine (Pcp), was tested for its antimicrobial efficacy against 103 strains belonging to both gram positive and gram negative bacteria. The drug was found to possess maximum activity against Staphylococcus aureus, Vibrio cholerae and Shigella spp. Pcp was moderately active against E. coli but most of the strains belonging to Bacillus spp, Klebsiella spp, Salmonella spp and Lactobacillus spp were found to be resistant to this drug. The drug was tested for its mode of antibacterial activity against Shigella dysenteriae 1 and it was found to be bacteriostatic in action. In in vivo studies, Pcp offered significant protection to Swiss albino mice at concentrations of 0.75 micro g/g (P < 0.01) and 1.5 microg/g (P < 0.001) body weight when challenged with 50 median lethal dose of Salmonella typhimurium NCTC 74. Thus the result depicts that prochlorperazine may emerge as a strong antimicrobial drug to replace the conventional antibiotics and to overcome the problem of drug resistance.     

Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface

AIMS: To investigate the biofilm formation by 122 Salmonella spp. and 48 Listeria monocytogenes strains on a plastic surface. METHODS: Quantification of biofilm formation was performed in brain heart infusion (BHI), trypcase soya broth (TSB), meat broth (MB) and 1/20 diluted trypcase soya broth (1/20-TSB) in plastic microtitre plates. RESULTS: All tested Salmonella spp. and L. monocytogenes strains produced biofilm in a suitable medium. However, the quantities of biofilm produced by Salmonella spp. were greater than those produced by tested L. monocytogenes strains. The nutrient content of the medium significantly influenced the quantity of produced biofilm. Diluted TSB was the most effective in promoting biofilm production by Salmonella spp., followed by TSB, while the least quantity of biofilm was formed in BHI and MB. L. monocytogenes produced the highest quantities of biofilm in BHI, followed by TSA, then MB, and the least quantities of biofilm were produced in 1/20-TSB. CONCLUSIONS: Salmonella spp. produces more biofilm in nutrient-poor medium, while L. monocytogenes produce more biofilm in nutrient-rich medium.     

Erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages

Aims:  To assess the frequency of erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages.
Methods and Results:  We isolated lactobacilli colonies from 20 salami from the north of Italy (Piacenza province) using selective medium supplemented with erythromycin or tetracycline; we determined the minimum inhibitory concentration and searched for selected erythromycin and tetracycline resistance genes. A total of 312 lactobacilli colonies were genetically ascribed to 60 different strains belonging to seven Lactobacillus species. Lactobacillus sakei , Lactobacillus curvatus and Lactobacillus plantarum were the most frequently found species. Thirty strains (50%) were phenotypically resistant to erythromycin, 45 (75%) to tetracycline and 27 (45%) were resistant to both. The most frequently detected resistance genes were tet (M) and erm (B).
Conclusions:  This study provides evidence of the presence of tetracycline- and, to a lesser extent, erythromycin-resistant lactobacilli in fermented dry sausages produced in northern Italy.
Significance and Impact of the Study:  Although these antibiotic-resistant lactobacilli could serve as reservoir organisms, in our study, 16 of 20 salami could be considered safe in regard to possible antibiotic resistance gene transfer to pathogens, whereas 4 of 20 could represent a borderline situation.     

Antibacterial activity of lactic acid bacteria isolated from vacuum-packaged meats

Lactic acid bacteria isolated from vacuum-packaged fresh meat stored at 4°C were shown to produce antagonistic substances active against closely related bacteria. Growth medium, pH and growth temperature all affected the production of the inhibitory substances. Ten strains including aciduric Lactobacillus -type organisms, Carnobacterium spp. and Leuconostoc spp. were selected that produced protein-aceous substances that caused inhibition of indicator strains. These were considered to be bacteriocins or bacteriocin-like compounds based on their inactivation with protease, generally narrow spectra of antibacterial activity and bactericidal or bacte-riostatic modes of action. Activity was not lost from supernatant fluids as a result of heat treatment at 62°C for 30 min, except for the Leuconostoc strains. Inhibitory spectra of some strains included Enterococcus spp. and Listeria monocytogenes . Some strains were of interest because their inhibitory substances were detected in the supernatant fluid early in the growth cycle. The inhibitory substances differed in characteristics between strains and there is evidence that more than one bacteriocin-like substance may be produced by some strains.     

多种食源性致病菌检测的多重PCR 方法的研究

目的:利用多重PCR技术,建立可以同时检测多种食源性致病菌的多重PCR方法。方法:分别选择沙门氏菌invA基因,志贺氏菌的ipaH基因,单核细胞增生李斯特氏菌的hlyA基因,大肠杆菌O157:H7的eaeA基因,副溶血弧菌的toxR基因,设计多重PCR引物,建立多重PCR检测体系,并对该体系进行特异性和灵敏度实验。结果:通过对19株菌株进行实验,所有的目标菌株均为阳性,而其余菌株为阴性。对多重PCR体系的灵敏度进行考察,沙门菌的灵敏度为5000 CFU/mL;志贺氏菌的灵敏度为5500CFU/mL;单核细胞增生李斯特氏菌的灵敏度为5200 CFU/mL;O157:H7的灵敏度为5000CFU/mL;副溶血弧菌的灵敏度为6300CFU/mL。结论:建立的多重PCR体系能实现多种致病菌同时检测。     

Microbial characterisation of fermented meat products from the Sicilian swine breed “Suino Nero Dei Nebrodi”

Two traditional sausage products (“salsiccia” and “salame”) processed from the raw meat of the Black Sicilian swine “Suino Nero dei Nebrodi” were microbiologically investigated during the manufacturing and ripening stages. Both products were dominated by lactic acid bacteria (LAB), especially rod-shaped types. The concentration of enterococci was consistent in salame. Coagulase-negative cocci increased slower than LAB. Yeasts showed an increasing trend during the ripening of both products. Enterobacteriaceae were counted at a constant level of about 10⁵ CFU/g in both products, while pseudomonads diminished during ripening. Coagulase-positive staphylococci, Listeria monocytogenes and Salmonella spp. were not detected at the end of the ripening process. Characterisation of LAB at the strain and species level revealed that Lactococcus lactis was found only in the meat mixture, while Lactobacillus sakei and various enterococci persisted during the monitoring period. Some LAB strains isolated from sausages were also identified on the surface of the factory equipment. Two strains (Lactobacillus sakei SS106A and Enterococcus faecalis SS91) were characterised by their anti-Listeria properties due to bacteriocin-like inhibitory substance production. A multiple strain starter composed of Lactobacillus sakei and enterococci has been proposed to maintain the typical characteristics of the two fermented meat products microbiologically investigated in this study.     

The microbial flora of smoked pork loin and frankfurter sausage stored in different gas atmospheres at 4 degrees C

The development of the microflora of smoked pork loin and frankfurter sausage was followed during storage in vacuum, N2 and CO2 atmospheres at 4 degrees C. The total aerobic count on the smoked pork loin reached 10(7) organisms/g after 37 d in vacuum, 43 d in N2 and 49 d in CO2. The corresponding value for the sausage was 77 d in vacuum, while the growth stopped at 6 x 10(40 organisms/g after 98 d in N2, and at 4 x 10(2) organisms/g after 48 d in CO2. The predominant organisms on the fresh products were Bacillus spp., coryneform bacteria, Flavobacterium spp. and Pseudomonas spp. At the end of the storage time the microflora on both products in the three gas atmospheres, consisted mainly of Lactobacillus spp. and two large groups of organisms that could not be identified as any described genus. Some of the unidentified strains could be classified as a Lactobacillus sp. after subsequent subculturing on laboratory media. The numbers of Lactobacillus spp. at the end of storage decreased in the order, CO2 greater than N2 greater than vacuum. Lactobacillus viridescens generally constituted a substantial part of the Lactobacillus flora (5-72%). On the sausages two large uniform groups of unidentifiable homofermentative Lactobacillus spp. were also found.     

Aggregation of lactobacilli and bifidobacteria with<Emphasis Type="Italic">Escherichia coli</Emphasis> O157

A total of 5 Bifidobacterium spp. isolated from pig and children' feces and 6 Lactobacillus spp. from chicken feces were examined for expression of aggregation, cell surface hydrophobicity (CSH) and adherence to intestinal mucin. Co-aggregation activity was seen in 3 strains of auto-aggregative bifidobacteria and 4 auto-aggregative strains of Lactobacillus spp. with 2 enterohemorrhagic E. coli (O157). CSH correlated with Lactobacillus auto-aggregating activity and adherence to mucin but the correlation between Bifidobacterium adherence to mucin and CSH was not confirmed.     

Lyophilized preparations of bacteriocinogenic Lactobacillus curvatus and Lactococcus lactis subsp. lactis as potential protective adjuncts to control Listeria monocytogenes in dry-fermented sausages

AIM: Study of the effectiveness of in situ bacteriocin production by lactic acid bacteria (LAB) to control Listeria monocytogenes in dry-fermented sausages. METHODS AND RESULTS: Two bacteriocin-producing strains: Lactococcus lactis subsp. lactis LMG21206 and Lactobacillus curvatus LBPE were grown in a pilot scale fermentor and lyophilized to be directly used in dry sausage fermentation. A commercial starter culture (Bel'meat SL-25) not inhibitory to L. monocytogenes (Bac- starter) was mixed (1 : 1) with each of the two lyophilized bacteriocin-producing strains to obtain starters active against the pathogen (Bac+ starter). Anti-Listeria effectiveness of the Bac+ starters was studied in dry-fermented sausages. The meat batter was experimentally contaminated with a mixture of four different strains of L. monocytogenes (10(2)-10(3) CFU g(-1)). The results showed that L. monocytogenes did not grow in any of the contaminated batches, but no significant decrease (P > 0.05) was observed either in the positive control (no added starter culture) or in samples fermented with the Bac- starter culture during the fermentation period and up to 15 days of drying. When the Bac+ starter contained Lb. curvatus LBPE, cell counts of L. monocytogenes decreased to below the detectable limit (<10 CFU g(-1)) after 4 h of fermentation and no survivors could be recovered by enrichment beyond day 8 of drying. When the Bac+ starter culture containing Lc. lactis LMG21206 was used, a decrease in Listeria counts to below the detectable limit was achieved after 15 days of drying. CONCLUSIONS: The bacteriocin-producing strains studied may be used as adjunct cultures for sausage fermentations to control the occurrence and survival of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Addition of the Bac+ strains, especially the Lb. curvatus strain would provide an additional hurdle to enhance the control of L. monocytogenes in fermented meat products.     

Adhesion of probiotic lactobacilli to chick intestinal mucus

In the present work, interactions between three Lactobacillus strains (Lactobacillus fermentum CRL1015, Lactobacillus animalis CRL1014, and Lactobacillus fermentum CRL1016) and chicken small intestinal mucus were determined. Three lactobacilli isolated from chicken and selected by their potentially probiotic properties were able to grow in mucus preparations. Three peaks from gel filtration chromatography of intestinal mucus were obtained. The adhesion to three mucus fractions (I, II, and III), especially fraction III, was higher (P < 0.01) in L. fermentum CRL1015 than L. animalis CRL1014. Pretreatment of this fraction with proteases and metaperiodate showed lower (P < 0.01) adhesion values than that of the control, suggesting that a glycoprotein from the mucus acts as a receptor for L. fermentum CRL1015. Highest adhesion values were obtained at pH 7 and 42 degrees C, and neither the removal of divalent cations with ethylenediaminetetraacetic acid (EDTA) nor the addition of calcium produced significant variation from the adhesion values of the control (P > 0.01). This adhesion was only inhibited by N-acetyl-glucosamine. Salmonella pullorum and Salmonella gallinarum showed high (P < 0.01) values of adhesion to chick intestinal mucus. The results obtained from assays of the inhibition of adherence of Salmonella spp. to mucus, immobilized in polystyrene tissue culture wells, indicated that the pathogen adhesion was not reduced by lactobacilli (P > 0.05) or their spent culture supernatants (P > 0.05), suggesting that these strains did not interfere with the binding sites for Salmonella spp. adhesion to the small intestinal mucus.