Comparative characterization of Listeria monocytogenes isolated from Portuguese farmhouse ewe's cheese and from humans

In order to investigate the possible relationships between Listeria monocytogenes strains isolated from farmhouse ewe's cheese and clinical strains collected, in partially overlapping dates, from the same geographical area in Portugal, a total of 109 isolates from seven ewe's cheese manufactures (n=94) and from humans (n=15) were characterized by serotyping, RAPD, PFGE and allelic analysis of the virulent actA gene. Serotyping indicated the presence of four different serovars: 1/2a, 1/2b, 1/2c and 4b. The 15 clinical isolates were either serovar 4b (86.7%) or serovar 1/2b (13.3%). Among the 94 isolates from cheese and related environments the serovars prevalence was 1/2a (1.1%), 1/2b (17.0%), 1/2c (12.8%) and, unexpectedly, 4b (69.1%). Based on results obtained with PFGE typing of the strains, 25 genotypes were identified, 10 from farmhouses and 15 from human cases. Isolates from serovars 1/2a and 1/2c were assigned to single genotypes, respectively. Within serovars 1/2b and 4b three and 20 genotypes were established, respectively. RAPD typing of the isolates rendered 18 types indicating the lack of accuracy of the primers used in strain differentiation within serovar 4b. The actA gene typing of the strains showed a prevalence of actA gene type I (90.4%) compared with the rest of the strains that were all actA gene type II (9.6%). In spite of the fact that all the farmhouses were completely independent, the distribution of L. monocytogenes genotypes, intra and inter cheese manufactures, was relatively homogeneous, suggesting the existence of resident strains. In contrast, among human isolates there was a great genetic diversity. There was no common genotype between L. monocytogenes implicated in the cases of listeriosis and these cheese-related isolates, suggesting the absence of a causal relationship.     

Stress response of Listeria monocytogenes isolated from cheese and other foods

The responses to pH and sodium chloride of four strains of Listeria monocytogenes isolated from Portuguese cheese, with a sodium chloride concentration of about 2% (w/v) and a pH value from 5.1 to 6.2, were studied. Two isolates from meat and two clinical isolates related to food-borne listeriosis, in which the implicated food product had about 2-3.5% (w/v) sodium chloride, also were studied. The effect of temperature on pH and sodium chloride sensitivity was also determined. The results show that natural isolates vary in response to these stresses and the data were often at variance with previously published data. Strains varied in sensitivity to low pH and to high sodium chloride concentration but the cheese isolates tended to be more resistant. A lower temperature was associated with a decrease in resistance to low pH and to sodium chloride. All strains showed an acid tolerance response induction when grown at pH 5.5 and although the time required for maximum induction of the response varied between strains, 2 h of acid adaptation, at least, was necessary which is longer than previously reported. Some strains showed an osmotolerance response after incubation in 3.5% (w/v) sodium chloride. Osmoadaptation, in addition to inducing an osmotolerance response, also induced cross-protection against acid shock conditions (pH 3.5). The acid tolerance response also induced a cross-protection against osmotic shock conditions (20% (w/v) sodium chloride). In some cases there was a relationship between the degree of resistance and adaptation, but usually the behaviour of a particular strain was independent of the conditions from which it was isolated.     

台州市食源性单核细胞增生李斯特菌分子特征研究

目的了解台州市食品中分离的单核细胞增生李斯特菌的血清型、毒力基因以及基因分型情况,建立食源性单核细胞增生李斯特菌的分子特征本底信息,为食源性疾病的防治提供技术支持。方法对近几年从食品中分离的37株单核细胞增生李斯特菌进行多重PCR血清分型、9种毒力基因(prf A、inl A、inl B、iap、fla A、hly A、plc B、mpl和act A)PCR检测、PFGE基因分型,用Bio Numerics 6.6软件对分型数据进行聚类分析。结果 37株食源性单核细胞增生李斯特菌的血清型以1/2a或3a型别为主;所有菌株均检出4种以上毒力基因,有15株菌携带所有9种毒力基因;37株菌经Apa I酶切PFGE分型后,共得到22种带型,每种带型包含1~5株不等,相似度区间为67%~100%。结论台州市食源性单核细胞增生李斯特菌存在致病流行风险,建立的指纹图谱数据库可为食源性疾病的防治提供技术支持。     

熟肉制品中单核细胞增生李斯特氏菌耐药及致病的遗传分析

目的 获得中国不同省份熟肉制品中的33株单核细胞增生李斯特氏菌(单增李斯特菌)的抗生素敏感性特征图谱,并运用全基因组测序对菌株进行耐药和致病的基因遗传分析.方法 采用微量肉汤稀释法对33株熟肉制品中的单增李斯特菌进行药敏测定,同时进行高精度框架图测序,基因组序列经组装后通过相应的生物信息学流程进行数据分析.结果 33株...     

Modeling survival of Listeria monocytogenes in the traditional Greek soft cheese Katiki

In the present work, survival of Listeria monocytogenes in the traditional Greek soft, spreadable cheese Katiki was studied throughout the shelf life of the product. Samples of finished cheese were inoculated with a cocktail of five L. monocytogenes strains (ca. 6 log CFU g(-1)) and stored at 5, 10, 15, and 20 degrees C. Acid-stress adaptation or cross-protection to the same stress was also investigated by inoculation of acid-adapted cells in the product. The results showed that pathogen survival was biphasic. Various mathematical equations (Geeraerd, Cerf, Albert-Mafart, Whiting, Zwietering, and Baranyi models) were fitted to the experimental data. A thorough statistical analysis was performed to choose the best model. The Geeraerd model was finally selected, and the results revealed no acid tolerance acquisition (no significant differences, P > 0.05, in the survival rates of the non-acid-adapted and acid-adapted cells). Secondary modeling (second-order polynomial with a(0) = 0.8453, a(1) = -0.0743, and a(2) = 0.0059) of the survival rate (of sensitive population), and other parameters that were similar at all temperatures (fraction of initial population in the major population = 99.98%, survival rate of resistant population = 0.10 day(-1), and initial population = 6.29 log CFU g(-1)), showed that survival of the pathogen was temperature dependent with bacterial cells surviving for a longer period of time at lower temperatures. Finally, the developed predictive model was successfully validated at two independent temperatures (12 and 17 degrees C). This study underlines the usefulness of predictive modeling as a tool for realistic estimation and control of L. monocytogenes risk in food products. Such data are also useful when conducting risk assessment studies.     

A comparison of two procedures for the isolation of Listeria monocytogenes from raw chickens and soft cheese

A cold enrichment method and a modified FDA procedure were compared for the isolation of Listeria monocytogenes from raw chickens and soft cheese. L. monocytogenes was isolated from a total of 23 of 222 cheese and 70 of 160 chicken samples by either one or both methods. Neither method alone yielded all isolates from the two food types. Only 12 cheese and 13 chicken samples were shown to be positive by both methods, although the serotypes isolated were not always identical. On some occasions one method yielded L. monocytogenes while the other produced a different Listeria sp. Reasons for differences in the performance of the two procedures and various points of technical interest are discussed.     

Prevalence,characterization, and genetic diversity of Listeria monocytogenes isolated from foods of animal origin in North East India

Listeria monocytogenes is a pathogenic microorganism infects man mostly through food. A total of 1615 samples of foods of animal origin and water were collected from retail meat shops of North-Eastern India and processed. Sixty-three (3.9%) samples were positive for L. monocytogenes. Animal origin foods showing the highest prevalence was chevon (9.8%) followed by beef (8.9%), chicken (8.5%), pork (2.8%) and milk (1.8%). The prevalence rate in water from retail meat shops was 10%. Recovered L. monocytogenes were distributed into 3 serogroups, of which 74.6% fit in to 1/2a, 3a serogroup, 17.5% to 1/2b, 3b and 7.9 % to 4b, 4d, 4e serogroups. Thirty-five isolates out of 63 possessed all the tested four virulence genes. RAPD- and ERIC -PCR based analyses jointly revealed a discriminative genetic profile for the L. monocytogenes. On the whole, the occurrence of L. monocytogenes in foods of animal origin of North Eastern India displays public health hazard.     

Characterization of Listeria monocytogenes isolated from retail foods

Listeria monocytogenes isolates recovered from retail ready-to-eat (RTE) meats, raw chickens and fresh produce were characterized by serogroup identification using PCR, genotyping using pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Five L. monocytogenes serogroups were identified. Of the 167 isolates 68 (41%) belonged to serogroup 1/2b, 3b; 53 (32%) belonged to serogroup 4b, 4d, 4e; 43 (26%) belonged to serogroup 1/2a, 3a; 2 (1.2%) belonged to serogroup 1/2c, 3c; and 1 (0.6%) belonged to serogroup 4a, 4c. PFGE generated 120 patterns which correlated well with PCR serogrouping. Most L. monocytogenes isolates were resistant to sulfonamide (73%) and some were resistant to tetracycline (8.4%) and ciprofloxacin (1.8%). Tetracycline resistance was conjugatively transferable and the tet(M) gene was identified in 14 tetracycline-resistant isolates as well as their transconjugants. These findings indicate that L. monocytogenes present in food were diverse, and that resistance to one or more antibiotics among these isolates was common. In addition, the presence of potential serotype 4b in all food categories is of public health concern, as serotype 4b has been the serotype most frequently associated with human listeriosis.     

Detection and characterization of Listeria monocytogenes in Sao Jorge (Portugal) cheese production

Listeria monocytogenes is a foodborne pathogen that can cause serious invasive disease in humans. Because human listeriosis cases have previously been linked to consumption of contaminated cheese, control of this pathogen throughout the cheese production chain is of particular concern. To understand the potential for L. monocytogenes transmission via São Jorge cheese, a Portuguese artisanal cheese variety that bears a Protected Denomination of Origin classification, 357 raw milk, curd, natural whey starter, and cheese samples representative of the production chain of this cheese were collected over one year and tested for the presence of L. monocytogenes and selected physicochemical parameters. Although neither L. monocytogenes nor other Listeria spp. were detected in whey, curd, or cheese samples, 2 of the 105 raw milk samples analyzed were positive for L. monocytogenes. These 2 raw milk isolates represented a ribotype that has previously been linked to multiple human listeriosis outbreaks and cases elsewhere, indicating the potential of these isolates to cause human listeriosis. On average, physicochemical parameters of São Jorge cheese ripened for 4 mo presented values that likely minimize the risk of L. monocytogenes outgrowth during ripening and storage (mean pH = 5.48; mean moisture = 37.79%; mean NaCl concentration = 4.73%). However, some cheese samples evaluated in this study were characterized by physicochemical parameters that may allow growth and survival of L. monocytogenes. Even though our results indicate that raw milk used for São Jorge cheese manufacture as well as finished products is rarely contaminated with L. monocytogenes, continued efforts to control the presence of this pathogen in the São Jorge cheese production chain are urged and are critical to ensure the safety of this product.     

Survival of Listeria monocytogenes during manufacture, ripening and storage of soft lactic cheese made from raw goat milk

Soft lactic cheeses were manufactured with raw goat milk inoculated with Listeria monocytogenes. The physico-chemical and microbiological characteristics of curds and cheeses were determined after each processing step as well as during ripening and refrigerated storage. The fate of Listeria monocytogenes was evaluated by enumeration on PALCAM agar and by a qualitative detection after a double selective enrichment procedure. The results showed that the physico-chemical and microbiological characteristics of lactic cheeses caused a decrease of Listeria monocytogenes counts. However, this decrease did not lead to the complete disappearance of the pathogen and Listeria monocytogenes was able to survive in soft lactic cheeses made with raw goat milk.     

Characterization of Listeria monocytogenes isolates from 50 small-scale Austrian cheese factories

One hundred eighty-one small-scale cheese factories (annual production < 100,000 kg) were tested for the presence of Listeria monocytogenes in cheese and smear samples from 1997 to 2000. In total, 2615 samples were drawn. Fifty (27.6%) of 181 enterprises yielded L. monocytogenes. From 14 of the cheese-making facilities, we obtained more than four L. monocytogenes isolates. A total of 182 mostly cheese- and smear-borne L. monocytogenes strains were characterized by serotyping and pulsed-field gel electrophoresis. In 12 of 14 cheese factories, over half of the L. monocytogenes isolates were genetically indistinguishable by pulsetype. On average, genetically indistinguishable isolates were recovered for 11.9 months. Regarding serotypes, 27.3% of the isolates were of serovar 4b. Inadequate personal hygiene could explain the high prevalence of serovar 4b isolates in small-scale cheesemaking facilities. Forty-two percent of the serovar 4b isolates recovered from epidemiologically unlinked facilities (in comparison to 40 and 29% of the 1/2a and 1/2b isolates, respectively) were genetically indistinguishable from at least one other isolate. Indistinguishable serovar 1/2a and 1/2b isolates belonged to five and six different pulsetypes, respectively, whereas serovar 4b isolates belonged to only two pulsetypes. This finding suggested a wide distribution of genetically homologous serovar 4b isolates among the facilities tested in our study.     

Analysis of PCR-based methods for characterization of Listeria monocytogenes strains isolated from different sources

Listeria monocytogenes strains, isolated from various sources (food, environment, and animals), were used to test different PCR-based methods to investigate their capability to define the strain origin. RAPD-PCR with three primers and the SAU-PCR method, in which the DNA was first digested with the Sau3A restriction endonuclease and then amplified with a primer designed on the restriction site, were carried out, and the profiles obtained were used to perform cluster analysis. Based on the cluster analysis of Listeria spp. strains, obtained from international collections, the coefficient of similarity was selected. The results obtained showed that the methods tested in the study gave different levels of differentiation between the strains tested. The RAPD protocol using the P1254 primer and the SAU-PCR gave appreciable results only for strains isolated from animals and from a food processing plant in two different periods of the year 2003. Better differentiation was observed using the RAPD-PCR with primer D8635. As a matter of fact, it was able to distinguish L. monocytogenes obtained from different species of animals, different food samples and strains from the same production plant isolated in different periods of the year. Also primer M13 gave positive results, but the coefficient of similarity to use had to be increased to 80%. On the basis of the results observed, RAPD-PCR with primers D8635 and M13 should be considered reliable tools for epidemiological investigations focusing on L. monocytogenes.     

A comparison of gene expression of Listeria monocytogenes in vitro and in the soft cheese Crescenza

This study focuses on the expression analysis of virulence and stress response genes (plcA, hly, iap and sigB) in 11 different strains of Listeria monocytogenes. The first step of this study considered an in vitro analysis in synthetic laboratory medium. According to statistical analysis, significant differences emerged for genes sigB and plcA. Subsequently, a soft cheese at two temperatures (4 and 12 °C) and for different times (24 and 48 h) was evaluated. Significant expression differences emerged for the condition at 12 °C after 48 h. No association could be observed between gene expression profile and origin of the different strains.     

食品中单增李斯特菌的血清分型及脉冲场凝胶电泳分型

对进口食品和国内食品中的单增李斯特菌进行血清学分型和分子分型研究,探讨不同来源菌株之间的遗传相关性及不同分型方法之间的联系。对分离的单增李斯特菌进行血清学分型和脉冲场凝胶电泳分型。69株单增李斯特菌分为4种血清型,主要血清型为1/2a(3a)型,脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)分型分为55种带型,主要型别为GX6A12.CN0018。食品中的单增李斯特菌分子型别呈现多态性,进口食品分离株和国内食品分离株之间无相关性。     

加工环境中单核细胞增生性李斯特菌耐药特征分析

目的分析在流通过程中加工环境来源的食源性李斯特菌耐药性及耐药基因携带情况,探讨加工环境对该菌的影响。方法收集2016~2017年分离自加工环境中的71株单核细胞增生性李斯特菌,利用微量肉汤稀释法对8种抗菌药物进行药物敏感性分析,并采用PCR鉴定11种耐药基因(tet A、tet M、tet S、erm A、erm B、erm C、mec A、aac(6’)-Ib、van A、van B、cfr)。结果 71株Lm对庆大霉素(11.27%)、氨苄西林(4.22%)、头孢他啶(98.59%)、环丙沙星(97.18%)、四环素(21.12%)、红霉素(15.49%)、林可霉素(92.96%)均具有不同程度的耐药性,对万古霉素敏感。其中以耐受3~6种抗菌药为主,耐药情况较为严重。耐药基因检测表明四环素类tet A,tet M为主要耐药基因,检出率高达50%以上,其次为红霉素类erm A、erm B、erm C,检出率约11%,氨基糖苷类aac(6’)-Ib的检出率为5.63%。结论加工环境单核细胞增生性李斯特菌的耐药性呈上升趋势,其耐药表型与耐药基因型并不完全一致,从而表明加工环境中存在影响单核细胞增生性李斯特菌耐药性因素,同时为食源性李斯特菌病的防治及预警提供参考依据。     

Molecular characterization of Listeria monocytogenes isolated from a poultry further processing facility and from fully cooked product

This study was undertaken to explore environmental sources of Listeria monocytogenes in a commercial chicken further processing facility and to compare the isolates obtained from this facility with others obtained from fully cooked product. In a survey conducted at the processing facility, 40 environmental sites (encompassing two production lines and representing areas in which raw and cooked products are processed) were cultured for L. monocytogenes. The resulting isolates were subjected to molecular subtyping by ribotyping, and these isolates were compared with 25 isolates collected by plant personnel from product contact surfaces and from fully cooked product. Eighty-nine environmental and product isolates were divided into 15 distinct ribogroups. Two ribogroups included isolates from fully cooked product; the members of these two ribogroups were subjected to further analysis by pulsed-field gel electrophoresis, resulting in four clusters. L. monocytogenes isolates from fully cooked product produced on line 1 were found to be indistinguishable from isolates collected from (i) drains on the raw-product side of line 1 and (ii) the floor surface in the cooked-product area of line 1. L. monocytogenes isolates from fully cooked product from line 2 were found to be indistinguishable from isolates collected from (i) the spiral freezer exit conveyor on line 2, (ii) raw product contact surfaces on line 1, and (iii) drains in the cooked-product area of line 1. These data suggest that L. monocytogenes can colonize a poultry further processing facility and eventually be transferred to fully cooked product.     

Prevalence and genetic characterization of Listeria monocytogenes in retail broiler meat in Estonia

The prevalence and genetic diversity of Listeria monocytogenes in raw broiler legs at the retail level in Estonia were studied. A total of 240 raw broiler legs (120 from Estonia and 120 of foreign origin, which had been imported to Estonia from Denmark, Finland, Hungary, Sweden, and the United States) from 12 retail stores in the two largest cities in Estonia (Tallinn and Tartu) were investigated from January to December 2002. Of these, 70% were positive for L. monocytogenes. The prevalence of L. monocytogenes in broiler legs of Estonian origin (88%) was significantly higher than in broiler legs of foreign origin (53%) (P < 0.001). Altogether, 169 (106 Estonian and 63 imported) L. monocytogenes isolates were characterized by pulsed-field gel electrophoresis (PFGE) typing after treatment with the restriction enzyme AscI. The isolates showed a wide genetic diversity, with 35 different PFGE types obtained. Of these, 11 PFGE types came only from isolates of broiler legs of Estonian origin, 4 of Danish origin, 2 of Finnish origin, and 4 of Hungarian origin. Fourteen PFGE types came from isolates of broiler legs that originated from various countries. The strains that shared the same PFGE types from isolates of Estonian origin were recovered from broiler legs that came from different stores over the course of several months. Seventy-one L. monocytogenes isolates, including all PFGE types, were serotyped, and three serotypes (1/2a, 1/2b, and 4b) were obtained. Serotype 1/2a accounted for 96% of the isolates.     

Prevalence and antimicrobial resistance of Listeria monocytogenes isolated in chicken slaughterhouses in Northern Greece

This study was conducted to determine the prevalence and antimicrobial resistance of Listeria monocytogenes recovered from chicken carcasses in slaughterhouses in Northern Greece. A total of 100 poultry samples (300 carcasses) were examined for Listeria spp. The samples were neck skin taken from four different slaughterhouses in Northern Greece. Forty samples were also taken from the environment of the slaughterhouses. Identification of L. monocytogenes was carried out by PCR and fingerprinting of the isolates by random amplified polymorphic DNA. L. monocytogenes strains isolated from chicken carcasses and from the environment of the slaughterhouses were also examined for antibiotic resistance. Fifty-five isolates of L. monocytogenes were tested for susceptibility to 20 antibiotics using the disk diffusion method. Listeria spp. were present in 99 of the poultry samples tested (99%), and 38 yielded L monocytogenes (38%). L. monocytogenes was also isolated in 80% of samples from the environment of a certain slaughterhouse, while the other slaughterhouses were found to be contaminated only with Listeria spp. All isolates were resistant to nalidixic acid and oxolinic acid, the majority of them to clindamycin, and only a few to tetracycline and oxytetracycline, whereas they were found to be susceptible to all other antimicrobials. The results of this study demonstrate a high prevalence of L. monocytogenes contamination in chicken carcasses, and all isolates were found to be sensitive to the antimicrobials most commonly used to treat human listeriosis.     

Survival of Listeria monocytogenes in commercial cheese brines.

The survival of Listeria monocytogenes was determined in commercial cheese brines collected from cheese factories in Wisconsin and northern Illinois. Survival of L. monocytogenes inoculated into commercial cheese brines ranged from < 7 d to over 259 d. Survival did not correlate with pH, salt content, nitrogen content, mineral content, or inherent microbial populations but was negatively associated with addition of sodium hypochlorite at the dairy plant. The L. monocytogenes generally survived longer in brines held at 4 degrees C than at 12 degrees C. Sodium hypochlorite or hydrogen peroxide inactivated L. monocytogenes when added to commercial brines in the lab at 10 to 100 ppm or 0.001% to 0.02%, respectively. Addition of 1% potassium sorbate or 1% sodium benzoate also decreased survival of L. monocytogenes. Laboratory filtration of commercial brines had a negative effect on survival in one of three brines tested. The L. monocytogenes did not grow during incubation in any of the commercial brine samples tested.