Listeria monocytogenes in Aquatic Food Products—A Review

With the increased demand for lightly preserved and/or ready‐to‐eat (RTE) food products, the prevalence of the foodborne pathogen Listeria monocytogenes has increased, which is a public health concern. The goal for this review is to discuss the incidence, epidemiological importance, and contamination routes of L. monocytogenes in various aquatic ecosystems, seafood products, and processing environments and to summarize the data obtained since the 1990s. L. monocytogenes primarily enters the food‐production chain by cross‐contamination in production plants, making this pathogen a major threat to the seafood industry. This pathogen generally contaminates food products at low or moderate levels, but the levels involved in listeriosis outbreaks are significantly higher. The majority of isolates from aquatic products belong to serotype 1/2a, and outbreaks have been linked to highly similar or even indistinguishable strains. Several seafood‐processing plants are colonized by specific “in‐house” flora containing special DNA subtypes of L. monocytogenes. In such cases, L. monocytogenes populations can persist and/or multiply despite the inherent obstacles to their growth in food preservation and manufacturing operations. Therefore, food‐processing facilities must be designed carefully with an emphasis on effective cleaning and disinfecting operations in the production line.     

Investigation of Listeria monocytogenes contamination pattern in local Chinese food market and the tracing of two clinical isolates by RAPD analysis

This study was undertaken to investigate the contamination pattern of Listeria monocytogenes in local Chinese food markets and to trace two clinical isolates. Random amplification polymorphic DNA (RAPD) was developed to track the source of L. monocytogenes in ready-to-eat (RTE) food products and from clinical origin. Three random primers, PB1, PB4 and HLWL74, were used to subtype all the L. monocytogenes strains isolated from RTE food products, fresh food products, environmental sewages in the markets and two clinical meningitis patients. It was shown that all the 49 isolates could be classified into 5, 4 and 4 types using these three random primers, PB1, PB4, and HLWL74, respectively. Twenty-seven composite profiles were identified by a combination of the three primers. The same composite profiles of L. monocytogenes could be found both in the fresh food products, environmental sewages and RTE food products, suggesting that the L. monocytogenes in the RTE food products may come from those in the fresh food products or sewage in the same market. The composite profiles of the two clinical isolates were the same as those of strains isolated from RTE food products, indicating that the disease might have resulted from the consumption of the RTE food products contaminated with L. monocytogenes. The results show that RAPD could be a powerful tool for the investigation of contamination pattern of L. monocytogenes in Chinese food markets and also for tracking the source of L. monocytogenes in clinical patients.     

Listeria monocytogenes: A Target for Bacteriophage Biocontrol

Listeria monocytogenes is a growing concern in the food industry as it is the causative agent of human listeriosis. There are many research articles concerning the growth, survival, and diversity of L. monocytogenes strains isolated from food‐related sources, elucidating the difficulty in controlling these bacteria in a food‐processing facility. Bacteriophage biocontrol of L. monocytogenes strains was introduced in 2006, through the first commercial bacteriophage product targeting L. monocytogenes ListShield^TM. This review focuses on the use of bacteriophage biocontrol to target L. monocytogenes in the food industry, specifically direct application of the bacteriophages to food products. In addition, we discuss characteristics of these bacteria that will have a significant influence on the effective treatment of bacteriophages such as genetic diversity between strains prevalent in one facility. There are many positive results of phage treatments targeting L. monocytogenes in food; however, success of in vitro studies might not be reproducible in practice. Future studies should focus on creating experimental design that will imitate the conditions found in the food industry, such as a stressed state of the targeted bacteria. In situ evaluation of bacteriophage treatment of L. monocytogenes will also be necessary because the presence of these bacteria in a processing facility can vary greatly regarding genetic diversity. The potential use of phages in the food‐processing facility as a biosanitizer for L. monocytogenes, as well as the use of lysins to target these bacteria should also be explored. Despite the exciting research avenues that have to be explored, current research shows that biocontrol of L. monocytogenes is feasible and has potential to positively impact the food industry.     

Listeria monocytogenes in retail establishments: Contamination routes and control strategies

In the past two decades, serious outbreaks of foodborne disease were caused by Listeria monocytogenes, a pathogen frequently found in delicatessens at retail. Although the prevalence of listeriosis is not high, the severity of disease is significant, with high hospitalization and mortality rates. Potential sources of L. monocytogenes and food contamination routes in retail and food service operations include incoming raw materials, food products, food handlers, customers, vendors, and environmental sources. Risk mitigation strategies for L. monocytogenes should be based on integrated control along the food chain continuum, from farm to retail establishment.     

中国食源性单核细胞增生李斯特菌耐药特征分析

目的研究中国22个省市自治区9类食品中分离的单核细胞增生李斯特菌的耐药特征。方法采用微量肉汤稀释法测定了1 069株单核细胞增生李斯特菌对庆大霉素、氨苄西林、青霉素、四环素、强力霉素、亚胺培南、红霉素、环丙沙星、左旋氧氟沙星、头孢噻吩、利福平、万古霉素、氯霉素、氨苄西林/舒巴坦酸和复方新诺明15种抗生素的耐药性。结果 1 069株单核细胞增生李斯特菌耐药率为6.92%,主要耐受的抗生素有四环素、强力霉素、红霉素、氯霉素和环丙沙星,并出现了多重耐药株。在不同食品来源中,分离自速冻米面制品的菌株耐药率最高,为9.64%。在22个省市自治区中,耐药率前3位的是:甘肃、吉林、福建,分别为27.3%、20.4%和17.4%。结论中国食源性单核细胞增生李斯特菌的耐药率较低,不同地区、不同食物来源耐药率差异较大,仍要加强对生产和临床使用抗生素的管理,减缓单核细胞增生李斯特菌耐药趋势的上升。     

Development of an Internal Amplification Control Using Multiplex PCR for the Detection of Listeria monocytogenes in Food Products

The bacterial pathogen Listeria monocytogenes is responsible for listeriosis, a food-borne disease, which may result in severe illness and possible death. Large outbreaks of listeriosis have been associated with food products including soft cheeses and ready to eat food products. Polymerase chain reaction (PCR) is a molecular identification method for food-borne pathogens; however, a drawback of this method is that false-positive or false-negative results may occur. To validate the accuracy of the PCR as a powerful molecular tool for pathogen detection, it is important that false-negative results be distinguishable from true-negative PCR results. The aim of this study was to design and include an internal amplification control (IAC) within the PCR to coamplify with L. monocytogenes in order to identify false-negative results of L. monocytogenes from ostrich meat and camembert cheese samples. The IAC had to be incorporated into the PCR without loss of specificity and sensitivity on the detection limit of L. monocytogenes and was developed and tested for use in a multiplex PCR detection system. A region of the pUC19 plasmid was selected as the IAC for this study. The optimal concentration at which pUC19 would coamplify with L. monocytogenes was determined to be 0.001 pg/μL. Following an enrichment procedure, the minimum number of organisms detected in a spiked food sample by the PCR was 8 CFU/mL L. monocytogenes; the same detection limit was attained when the pUC19 IAC was included in the PCR. An optimal pUC19 IAC concentration increased the reliability of the PCR for food diagnostic purposes.     

蜂粮中Lactobacillus kunkeei的益生特性研究

蜂粮是植物花粉、蜂蜜和蜜蜂唾液的发酵混合物,是营养丰富的天然食材。作者从蜂粮中分离到61株乳酸杆菌,经16S rRNA基因测序,鉴定为Lactobacillus kunkeei。首先,用邻苯二甲醛法对61株L.kunkeei进行胆固醇去除能力检测,其中5株菌胆固醇去除能力均大于15%,菌株B35对胆固醇的去除率达到(29.07±1.30)%。随后,对这5株L.kunkeei进行酸耐受性、胆盐耐受性、抗生素耐药性及抑菌等益生特性进行研究。耐酸实验和耐胆盐实验结果表明,5株L.kunkeei均具有良好的耐酸性和胆盐耐受性。药敏试验结果表明,5株L.kunkeei对4种临床常用抗生素(红霉素、克林霉素、庆大霉素、万古霉素)均敏感。通过琼脂扩散法抑菌实验,发现5株L.kunkeei对大肠埃希氏菌、金黄色葡萄球菌均有一定的抑菌作用。B35菌株的细胞黏附性能良好,对Caco-2细胞的黏附率达到3.32%。从蜂粮中分离到5株具有良好益生特性的菌株,其中菌株B35的益生特性最佳,为益生菌的开发与利用提供了新的菌种资源。     

A method based on the ligation detection reaction–universal array (LDR–UA) for the detection and characterization of Listeria and Campylobacter strains

Listeria and Campylobacter genera include some of the most widely spread human pathogens across Europe and represent a serious health threat, especially to children, immunocompromised people and pregnant women. Both genera are frequently isolated from farm animals and food; therefore, their rapid detection is important for food safety and to prevent disease outbreaks. A rapid detection approach based on the combination of ligation detection reaction and universal array (LDR–UA) was developed to reveal the presence of Listeria and Campylobacter pathogenic species and to identify the Division (I, II and III) of L. monocytogenes isolates. The approach was tested first on reference strains then on field isolates. The LDR–UA approach showed high sensitivity and high specificity in reliably discriminate target sequences differing in as little as one base pair, thus facilitating the discrimination of closely related strains.     

Listeria monocytogenes: Strain Heterogeneity,Methods, and Challenges of Subtyping

Listeria monocytogenes is a food‐borne bacterial pathogen that is associated with 20% to 30% case fatality rate. L. monocytogenes is a genetically heterogeneous species, with a small fraction of strains (serotypes 1/2a, 1/2b, 4b) implicated in human listeriosis. Monitoring and source tracking of L. monocytogenes involve the use of subtyping methods, with the performance of genetic‐based methods found to be superior to phenotypic‐based ones. Various methods have been used to subtype L. monocytogenes isolates, with the pulsed‐field gel electrophoresis (PFGE) being the gold standard. Although PFGE has had a massive impact on food safety through the establishment of the PulseNet, there is no doubt that whole genome sequence (WGS) typing is accurate, has a discriminatory power superior to any known method, and allows genome‐wide differences between strains to be quantified through the comparison of nucleotide sequences. This review focuses on the different techniques that have been used to type L. monocytogenes strains, their performance challenges, and the tremendous impact WGS typing could have on the food safety landscape.     

Antibiotic-Resistant Bacteria: A Challenge for the Food Industry

Antibiotic-resistant bacteria were first described in the 1940s, but whereas new antibiotics were being discovered at a steady rate, the consequences of this phenomenon were slow to be appreciated. At present, the paucity of new antimicrobials coming into the market has led to the problem of antibiotic resistance fast escalating into a global health crisis. Although the selective pressure exerted by the use of antibiotics (particularly overuse or misuse) has been deemed the major factor in the emergence of bacterial resistance to these antimicrobials, concerns about the role of the food industry have been growing in recent years and have been raised at both national and international levels. The selective pressure exerted by the use of antibiotics (primary production) and biocides (e.g., disinfectants, food and feed preservatives, or decontaminants) is the main driving force behind the selection and spread of antimicrobial resistance throughout the food chain. Genetically modified (GM) crops with antibiotic resistance marker genes, microorganisms added intentionally to the food chain (probiotic or technological) with potentially transferable antimicrobial resistance genes, and food processing technologies used at sub-lethal doses (e.g., alternative non-thermal treatments) are also issues for concern. This paper presents the main trends in antibiotic resistance and antibiotic development in recent decades, as well as their economic and health consequences, current knowledge concerning the generation, dissemination, and mechanisms of antibacterial resistance, progress to date on the possible routes for emergence of resistance throughout the food chain and the role of foods as a vehicle for antibiotic-resistant bacteria. The main approaches to prevention and control of the development, selection, and spread of antibacterial resistance in the food industry are also addressed.     

Prevalence and characterization of antimicrobial resistance of foodborne Listeria monocytogenes isolates in Hebei province of Northern China, 2005-2007

A total of 2177 food samples collected from nine cities in northern China during 2005 to 2007 were screened for the presence of Listeria monocytogenes. All L. monocytogenes isolates were subjected to serotyping, antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), as well as PCR screening to identify genes responsible for tetracycline resistance [tet(L), tet(M), tet(K), tet(S) and tet(B)], transposon Tn916, and class 1 integron. Contamination with L. monocytogenes was detected in 4.13% (90/2177) of the total samples representing various food products. The pathogen was mainly isolated from frozen food made of wheat flour or rice products (26/252, 10.32%) and raw meat products (46/733, 6.28%). Besides, 3.31% (10/302) of cooked meat, 1.17% (4/343) of seafood, 0.98% (2/204) of non-fermented bean products and 0.62% (2/323) of vegetables samples were contaminated by this bacterium. The L. monocytogenes isolates belonged to five serotypes (1/2a, 1/2b, 1/2c, 4b, and 3a), with serotype 1/2a being dominant (48.88%). Antimicrobial resistance was most frequently observed for ciprofloxacin (17.8%), tetracycline (15.6%) and streptomycin (12.2%). Overall, resistance was observed against 14 out of 18 antimicrobials tested while multiple resistances occurred among 18.9% (17/90) isolates. Interestingly, two isolates were resistant to more than five antimicrobials. Among 14 tetracycline-resistant isolates, 13 carried tet(M) gene including nine possessing Tn916, and one harbored tet(S) gene. PFGE analysis revealed genetic heterogeneity among individual serotypes as well as scattered occurrence of some genotypes without any clear-cut correlation to source or food type. The widespread distribution of epidemiologically important serotypes (1/2a, 1/2b and 4b) of L. monocytogenes, and their resistance to commonly used antibiotics indicate a potential public health risk. Our data also indicate that L. monocytogenes could act as a reservoir of mobile tet genes along the food chain.     

Comparison of two AFLP methods and PFGE using strains of Listeria monocytogenes isolated from environmental and food samples obtained from Piedmont, Italy

Listeria monocytogenes ranks among the most frequent causes of death due to foodborne illness (20-30% case fatality rate).Discriminative subtyping methods are important to detect the relatedness of isolates and verify epidemiologic associations. AFLP analysis is a DNA fingerprinting technique based on the selective amplification of genomic restriction fragments. In this study, two AFLP methods and PFGE were compared in regard to discriminatory power, typeability and concordance.A total of 103 unrelated L. monocytogenes strains isolated from different environmental and food sources were analyzed. Strains were isolated from samples obtained from food-production plants, supermarkets and small food markets in Piedmont, Italy.All methods clustered L. monocytogenes strains into two genetic lineages, Lineage I and II. The three methods were compared using the 82 isolates which were typeable with all techniques. The calculated pair-wise Pearson's correlation coefficients (r) showed close agreement between all three methods.Our findings suggest that the AFLP II method can be successfully used to subtype L. monocytogenes strains isolated from foods and food processing facilities.     

Enumeration of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> at Low Contamination Levels in Several Food Matrices Using a Membrane Filtration Method

For the enumeration of Listeria monocytogenes in cold-smoked salmon, a sensitive enumeration method, based on membrane filtration followed by transfer of the filter on a selective medium, has been recently developed. An evaluation of this method was performed with several categories of foods likely to be contaminated with L. monocytogenes. The results obtained with the technique were compared with those from the reference EN ISO 11290-2 method and found to provide more precise results in the enumeration of L. monocytogenes from both artificially and naturally contaminated products. In most cases, the filtration method enabled a greater quantity of food to be examined (from around 0.5 to 14 g, instead of 0.01 to 0.1 g with the reference EN ISO 11290-2 method), thus greatly improving the sensitivity of the enumeration.     

陕西省常见食源性致病菌耐药性研究进展

近年来食品安全问题层出不穷,而食源性致病菌则是引起食品安全问题的主要因素之一,严重危害了人类的健康。引发食源性疾病的常见致病菌主要有大肠杆菌、沙门氏菌、金黄色葡萄球菌、单核细胞增生李斯特菌等。研究表明,在过去的几十年里,由于在医疗、养殖业等领域中过度使用抗生素,造成细菌耐药现象日趋严重,这更加重了食源性致病菌的潜在危险。尽管食源性致病菌的耐药性在国家层面有监测网,但是聚焦省级地区仍是以点代面。作者旨在通过综述分析陕西省的食源性致病菌的耐药现状,以期为食源性致病菌耐药性监测提供依据。     

Prevalence, characterization and sources of Listeria monocytogenes in blue crab (Callinectus sapidus) meat and blue crab processing plants

Seven blue crab processing plants were sampled to determine the prevalence and sources of Listeria spp. and Listeria monocytogenes for two years (2006–2007). A total of 488 raw crabs, 624 cooked crab meat (crab meat) and 624 environmental samples were tested by standard methods. Presumptive Listeria spp. were isolated from 19.5% of raw crabs, 10.8% of crab meat, and 69.5% of environmental samples. L. monocytogenes was isolated from 4.5% of raw crabs, 0.2% of crab meat, and 2.1% of environmental samples. Ninety-seven percent of the isolates were resistant to at least one of the ten antibiotics tested. Eight different serotypes were found among 76 L. monocytogenes isolates tested with the most common being 4b, 1/2b and 1/2a. Automated EcoRI ribotyping differentiated 11 ribotypes among the 106 L. monocytogenes isolates. Based on ribotyping analysis, the distribution of the ribotypes in each processing plant had a unique contamination pattern. A total of 92 ApaI and 88 AscI pulsotypes among the 106 L. monocytogenes isolates were found and distinct pulsotypes were observed in raw crab, crab meat and environmental samples. Ribotypes and serotypes recovered from crab processing plants included subtypes that have been associated with listeriosis cases in other food outbreaks. Our findings suggest that molecular methods may provide critical information about sources of L. monocytogenes in crab processing plants and will augment efforts to improve food safety control strategies such as targeting specific sources of contamination and use of aggressive detergents prior to sanitizing.     

The occurrence of Listeria monocytogenes in cheese from a manufacturer associated with a case of listeriosis

A case of listeriosis was associated with the consumption of a soft cheese produced in England. Goats cheese and other products from the same food manufacturer were examined for the presence of Listeria over the following 11 months. Listeria monocytogenes was isolated from 16 of 25 cheese samples on retail sale, 12 of 24 cheese samples obtained directly from the factory, and from shelving within the plant. Phage-typing of 68 isolates of L. monocytogenes from cheese samples and the factory showed that 66 (97%) were indistinguishable from the strain isolated from the patient's cerebrospinal fluid and stool. L. monocytogenes was not isolated from seven goats milk or two yoghurt samples. Listeria innocua was isolated from 10 cheese samples, two of which contained no other species of Listeria. Levels of L. monocytogenes shortly after production were low (<10/g), but were higher (10⁵–10⁷ cfu/g) in six of the 16 cheese samples obtained from retail outlets. Multiplication of L. monocytogenes was demonstrated in cheeses contaminated at the factory and held at 4°C in the laboratory.     

Occurrence and characterization of enterotoxigenic Staphylococcus aureus isolated from minimally processed vegetables and sprouts in Korea

Staphylococcus aureus has long been recognized as an important pathogen in food-borne disease in the world. Minimally processed vegetables and sprouts are often contaminated with enterotoxigenic strains of this bacterium. This paper reports the results of a 3-year survey (2006–2008) on the occurrence of S. aureus in minimally processed vegetables and sprouts. Of 345 examined samples, 40 samples (11.6%) were contaminated with S. aureus. A total of 25 enterotoxigenic S. aureus strains were biotyped and their resistance to antibiotics was examined. Most isolated strains produced Staphylococcal enterotoxin A (SEA) (n=23) followed by Staphylococcal enterotoxin I (SEI) and Staphylococcal enterotoxin G (SEG) and mainly belonged to the human biotype (88%). At least 96.1% of the analyzed strains showed antibiotic resistance properties, while 56% of the analyzed strains exhibited multiple antibiotic resistance to the antibiotics tested. Two of the analyzed strains were resistant to methicillin. Moreover, a strain which had multi-resistance to 6 antibiotics was found. The results indicate that enterotoxigenic, antibioticresistant strains of S. aureus are widely proliferated in minimally processed vegetables and sprouts.     

Listeria species in seafood: isolation and characterization of Listeria spp. from seafood in San Luis, Argentina

One hundred seafood samples (fish, squid and mussel) were collected from the Argentine Atlantic coast and screened for Listeria spp. The isolates were characterized by biochemical tests, serotyping, phage typing and macrorestriction enzymes analysis of DNA (pulsed-field gel electrophoresis). The overall frecuency (n=100) ofListeria spp. was 12%. Of the 12 isolated strains, three strains isolated from different squid samples were identified as L. monocytogenes and nine strains from fish, mussels and squid as L. innocua. All three L. monocytogenes strains belonged to serovar 4b; eight strains of L. innocua were serovar 6a and one strain of L. innocua was serovar 6b. All three L. monocytogenes strains, but only one of the nine L. innocua strains, were phage-typeable. One restriction profile was detected with Apa I and Asc I for L. monocytogenes strains and three with the same enzymes for L. innocua strains. The combination of these patterns allowed definition of four distinct groups within the 12 strains. The results of this study showed that phenotypic methods remain appropriate but that pulsed-field gel electrophoresis is useful for epidemiological purposes.     

Reliability of Listeria monocytogenes Identification by Specific PCR Assessed by Phenotypic and Genotypic Techniques

This study evaluates the possibility of using polymerase chain reaction (PCR) for rapid identification of food-borne Listeria monocytogenes as an alternative to API Listeria system and estimates the incidence of API Listeria misidentifications in food-borne Listeria species. A total of 198 strains, 11 L. monocytogenes, 28 other Listeria species, and 159 food isolates were phenotypically and genotypically characterized by API Listeria profiles and randomly amplified polymorphic DNA (RAPD) profiles, respectively. They were also tested for PCR amplification using genus- and species-specific primers. Clustering analysis of phenotypic and genotypic data showed discrepancies in species identification of some isolates by API Listeria profiles. Their identities were confirmed by 16S rDNA sequencing, and thus, it was revealed that 33% of Listeria innocua and 19% of Listeria welshimeri were misidentified as L. monocytogenes by API Listeria profiles. Reliable identification of L. monocytogenes was obtained by LM1–LM2 specific primers which allowed PCR amplification only in reference strains and isolates previously identified as L. monocytogenes by RAPD and 16S rDNA sequence analysis. These results corroborate the suitability of specific PCR as a rapid and accurate test for the identification of L. monocytogenes, avoiding misidentification with other Listeria species commonly found in food products.     

Safety and functional aspects of pre‐selected pediococci for probiotic use in Iberian dry‐fermented sausages

The purpose of this study was to investigate pre‐selected pediococci for potential probiotic use in Iberian dry‐fermented sausages. A total of twelve strains isolated from Iberian dry‐fermented sausages and pig faeces were evaluated according to safety and functional characteristics including biogenic amines and d ‐lactic acid production, antibiotic susceptibility, cell adhesion and antimicrobial activity against food‐borne pathogens. The strain P. acidilactici SP979 was able to establish itself and compete with enteropathogens such as Salmonella choleraesuis on the intestinal epithelium and an inhibition of such pathogenic bacteria as Listeria monocytogenes and Staphylococcus aureus in vitro. This strain was also considered safe to be used with regard to its void aminogenic potential, low d ‐lactic acid production and antibiotic resistance pattern; being identified as a potential probiotic meat starter culture suitable for manufacture of dry‐fermented Iberian sausages.