Shikonin-loaded bioactive gelatin films with antibacterial activity against Listeria species and antioxidant properties to extend the shelf-life of raw refrigerated beef.

PurposeListeriosis is one of the globally distributed foodborne diseases with the highest fatality rate. The objectives of this study were to isolate and identify Listeria species, assess factors for contamination of beef, and antibiogram of Listeria monocytogenes in Ambo and Holeta towns, Central Ethiopia.Materials and MethodsA total of 450 meat samples were collected from abattoirs (n=150), butchers (n=150), and restaurants (n=150) for isolation and identification of Listeria species. Logistic regression analysis was used to assess the association between the occurrence of Listeria species in meat and potential risk factors. The antimicrobial susceptibility test was done using the Kirby Bauer test.ResultsThe overall occurrence of Listeria species in Ambo and Holeta towns was 28.4% (128/450; 95% confidence interval [CI]: 24.3–32.9%). The isolation rate of Listeria monocytogenes was 4.4%, Listeria ivanovii 2.2%, Listeria seeligeri 1.8%, Listeria welshimeri 3.8%, Listeria innocua 6.2%, and Listeria grayi 10.2%. The probability of contamination of meat in butchers and restaurants was higher in Holeta than Ambo [OR=3.4; 95%; p=0.001], in dry than wet season [OR=5.2; p=0.009], and where the hygiene of cutting boards was poor (OR=7.7; p=0.008). Of the 20 Listeria monocytogenes isolates, 80%, 70%, 60%, and 55% were resistant to oxacillin, amikacin, and nalidixic acid, chloramphenicol, and tetracycline, respectively. The Listeria monocytogenes isolates were 95%, 90%, and 85% susceptible to amoxicillin, vancomycin, and clindamycin, respectively. About 95% of Listeria monocytogenes isolates were multidrug-resistant. One isolate (5%) had developed resistance to 10 classes of antimicrobial drugs.ConclusionListeria species are widespread and study towns, season, and hygiene of cutting boards are independent predictors of isolation of Listeria species. Multidrug resistance among Listeria monocytogenes was very high. Therefore, adequate cooking of meat, regular training of beef handlers, prudent use of drugs, and further molecular studies on Listeria species are important.

Rapid detection and differentiation of Listeria monocytogenes and Listeria species in deli meats by a new multiplex PCR method

This study was carried out in order to determine the disinfectant effect of Methylated spirit® (95% methanol and 5% ethanol) as a teat dip against Listeria species. Hand milking was employed to collect 576 (288 x 2) raw milk samples from different lactating cows within Sokoto metropolis (Nigeria). 288 samples were collected before disinfecting the udder teats with Methylated spirit®, while the other 288 were collected after disinfection with Methylated spirit®. The samples were analyzed using selective culture and isolation technique in which the 288 samples collected before disinfection, 114 (39.6%) were positive for Listeria species. Among the positive samples 44 (38.6%) were Listeria innocua, 16 (14.0%) Listeria ivanovii, 36 (31.6%) Listeria monocytogenes, 11 (9.6%) Listeria welshimeri and 7 (6.1%) Listeria seeligeri, while none of the 288 samples collected after disinfection was positive. The study has shown high prevalence of Listeria species in milk collected without washing/disinfecting the teats and has also established the sensitivity of Listeria species to methylated ethanol which can be used as dip for disinfecting udder teats before milking in order to prevent contamination with Listeria species and other methylated spirit-sensitive organisms. This study is essential to educate Fulani herdsmen and other milk handlers on the importance of disinfecting udder teats before milking.

Most known Listeria monocytogenes virulence genes cluster within a 9.6-kb chromosomal region. This region is flanked on one end by two uncharacterized open reading frames (ORF A and ORF B) and ldh, an ORF presumably encoding the L. monocytogenes lactate dehydrogenase (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). We report here that the other end is flanked by prs, and ORF homologous to phosphoribosyl PPi synthetase genes. ORF B and prs were detected in all Listeria species and thus delimit the virulence region. This virulence gene cluster was detected exclusively in hemolytic Listeria species, Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species.

Background: Listeria monocytogenes, a member of the genus Listeria, is widely distributed in agricultural environments, such as soil, manure and water. The genus of Listeria bacteria is about 15-17 species. It is a pathogenic bacterium that can cause a rare but dangerous infection called listeriosis.
 Objectives: Studying the rate of salads contaminated with Listeria bacteria. and Listeria monocytogenes according to International, Arabic and Iraqi specifications and finding the correlation between commitments of restaurants to standard health conditions with contamination with these bacteria
 Methods: The study included 152 samples of salads taken from 39 restaurants chosen randomly and of different levels and places in Baghdad from the period between 1/9/2014 to 20/1/2015. The laboratory tests were carried out on samples based on internationally approved methods in addition to methods of the International Standards Organization.
 Results: The study revealed that 23 samples (15.13%) from the 152 samples taken from the restaurants were contaminated with Listeria species. of these, 3 (2%) were contaminated with Listeria monocytogenes and 20 (13.2%) were contaminated with other types of different and non-pathogenic Listeria as follows; (Listeria welshimeri, Listeria seeligeri, Listeria ivanovii, Listeria grayi, Listeria innocua) with the following prevalence (7(4.6%), 6(3.9%), 3(2%), 3(2%), 1 (0.7%) respectively).
 Conclusions: Contamination of salads taken from restaurants with Listeria bacteria is not uncommon. This indicates that routine examination is necessary and should be added to the Iraqi standard for salads.

Simultaneous detection of Listeria species isolated from meat processed foods using multiplex PCR

A gene, designated msp, that encodes a major secreted polypeptide with a molecular mass of approximately 60 kilodaltons (kDa) was cloned from Listeria monocytogenes 10403. DNA hybridization analysis indicated that the msp gene was highly conserved among 15 independent L. monocytogenes isolates and that each of 5 isolates tested secreted a 60-kDa polypeptide that was immunologically related to the msp gene product. DNA sequences related to msp were not detected in any other Listeria species or in strains of Bacillus cereus, Bacillus thuringiensis, Streptococcus pyogenes, or Streptococcus pneumoniae when standard stringent DNA hybridization conditions were used. Under nonstringent conditions, related sequences were detected in Listeria ivanovii, Listeria seeligeri, and Listeria innocua, and immunoblot analysis indicated that these strains secreted polypeptides of about 60 kDa that were immunologically related to the msp gene product. The possibility of using the msp gene as a probe for the detection of L. monocytogenes and the potential functions of the msp gene product are discussed.

Listeria Species in Raw and Ready-to-Eat Foods from Restaurants

In a study designed to gain data on the in vitro transferability of vancomycin resistance from enterococci of the VanA phenotype to listeriae of different species, three clinical Enterococcus isolates-Enterococcus faecium LS10, Enterococcus faecalis LS4, and Enterococcus faecalis A3208, all harboring a plasmid that strongly hybridized with a vanA probe-were used as donors in transfer experiments. Strains of five Listeria species were used as recipients. From Enterococcus faecium LS10, glycopeptide resistance was transferred to Listeria monocytogenes, Listeria ivanovii, and Listeria welshimeri recipients, whereas no transfer occurred to Listeria seeligeri or Listeria innocua strains. From the two Enterococcus faecalis isolates, no transfer occurred to any Listeria recipient. MICs of both vancomycin and teicoplanin were > or = 256 mg/l for all transconjugants tested. Furthermore, all transconjugants harbored a plasmid that strongly hybridized with the vanA probe, with vanA consistently located in an EcoRI fragment of about 4 kb. Exposure of Listeria transconjugants to vancomycin resulted in synthesis of a membrane protein similar in size (39 kDa) to a vancomycin-induced membrane protein of Enterococcus faecium LS10. In retransfer experiments with Listeria transconjugants used as donors, glycopeptide resistance was transferred to all Listeria recipients tested, including strains of Listeria innocua and Listeria seeligeri, which were unable to receive the resistance from Enterococcus faecium LS10. The frequency of vanA transfer to listerial recipients was greater in retransfer experiments than in the primary matings. These findings suggest that the vanA resistance determinant might spread to the established pathogen Listeria monocytogenes, both directly from a resistant enterococcus and through strains of nonpathogenic Listeria species acting as intermediate resistance vehicles.

Occurrence of Listeria monocytogenes and Other Listeria spp. in Raw Caprine Milk

Listeria in ready-to-eat and unprocessed foods produced in Portugal

Background : Listeria is one of the food-borne pathogens common contaminant in dairy products. Consumption of milk and dairy products, in particular soft cheese often implicated as the source of infection in severe outbreaks of listeriosis. Objective : This work aimed to investigate the prevalence of Listeria monocytogenes and other Listeria spp. in raw milk and white soft cheese by conventional methods and polymerase chain reaction. Methodology : Two hundreds samples (50 of each market raw milk, individual farm milk and Kariesh cheese plus 25 of each Damietta cheese and Talaga cheese) where randomly collected from dairy farms, different shops and supermarkets in Mansoura City, Dakahlia governorate. Direct isolation of Listeria on Oxford media was performed and compared with indirect (enrichment) method followed by biochemical identification. Polymerase chain reaction was done for accurate detection of Listeria monocytogenes. Results : The prevalence of Listeria monocytogenes by direct method was 10%, 8%, 8% and 4% in market raw milk, Kariesh cheese, Talaga cheese and Damietta cheese, respectively. On the other hand, indirect (enrichment) isolation of Listeria monocytogenes showed prevalence of 8% and 4% in market raw milk and Talaga cheese, respectively. Listeria ivanovii and Listeria Seeligeri were also detected as 24% and 12% in market raw milk, 24% and 30% in individual farm milk, 16% and 32% in Kariesh cheese, 48% and 36% in Talaga cheese and 24% and 36% in Damietta cheese by direct method. On the other hand, by indirect (enrichment) method the prevalence of Listeria ivanovii and Listeria seeligeri was 14% and 2% in market raw milk, 6% and 14% in individual farm milk, 12% and 12% in Talaga cheese and 12% and 16% in Damietta cheese, but Kariesh cheese was free form Listeria ivanovii and Listeria seeligeri was 4%. Conclusions : The general principles of food hygiene should still be enforced in order to minimize count of Listeria monocytogenes in milk and dairy products during the handling, storage and manufacturing in traditional dairies. Control of the feeding cattle and milk pasteurization can also limit the contamination with Listeria monocytogenes.

Objective: To analyze distribution of Listeria monocytogenes serotypes and antimicrobial susceptibility of Listeria isolates from a domestic swine processing facility. Materials and methods: Presumptive Listeria isolates (314) were molecularly identified to discriminate among L monocytogenes, Listeria ivanovii, and Listeria species. Listeria monocytogenes serotypes were identified by polymerase chain reaction (PCR) and PCR-restriction enzyme analysis (PCR-REA) and tested for antimicrobial susceptibility. Results: Isolates were identified as L monocytogenes (259; 82.5%), L ivanovii (2; 0.6%), and Listeria species (53; 16.9%). Distribution of L monocytogenes serotypes: 4a/4c (0.4%), 4b (11.2%), 4d/4e (14%), 4b/4d/4e (9.3%), 1/2a (26.3%), 3a (7.7%), 1/2a/3a (6.2%), 1/2b/3b (1.2%), 1/2c (5%), 3c (1.2%), and 1/2c/3c (5.4%). Thirty-two L monocytogenes isolates (12.4%) were not typeable by PCR-REA, suggesting the possibility of serotypes 4ab/7. Susceptibility was 84.2% to 100% for most antimicrobials. Major resistance (R) and intermediate (I) susceptibility were found for clindamycin (R = 36.7%, I = 39.8% for L monocytogenes; R = 100% for L ivanovii; and R = 14%, I = 86% for Listeria species). Drugs of choice for treatment of human listeriosis (penicillin, ampicillin, and trimethoprim-sulfamethoxazole) remained effective; 1.2% of L monocytogenes were β-lactam resistant. Multidrug resistance was found only in L monocytogenes (26.6%) and Listeria species (26.4%), with (clindamycinI or R -erythromycinR-azithromycinR) and (ciprofloxacinI-clindamycinI) the most frequent phenotypes. Implications: Resistance to clindamycin and ciprofloxacin are shared between L monocytogenes and untyped Listeria. Although erythromycin is a drug of choice for prophylaxis in Colombian swine, resistance is low. No specific relationships between serotypes, sources, and antimicrobial susceptibility were found.

In order to establish the taxonomic value of 16S rRNA and 23S rRNA for distinguishing Listeria species, the complete 23S rRNA sequences for all Listeria species were determined by using the type strains. We designed and experimentally validated a universal 23S rRNA sequencing method, which included PCR amplification of the rDNA gene and direct cycle sequencing of the amplicon with eubacterial primers. The results of our sequence comparison indicated that the genus Listeria can be divided into two subgroups; one subgroup is composed of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri, whereas the other subgroup includes Listeria grayi subsp. grayi and Listeria grayi subsp. murrayi. A phylogenetic analysis revealed that these species diverged recently. These results are consistent with 16S rRNA sequence analysis data. For application purposes, one 16S rRNA region that can be sued to distinguish each Listeria species except L. Monocytogenes and L. innocua has been described. In this study we found four 23S rRNA signature regions which, when used in combination, can be used to distinguish the species.

Aptamer-based magnetic isolation and specific detection system for Listeria monocytogenes from food samples