Effect of surface roughness and stainless steel finish on Listeria monocytogenes attachment and biofilm formation

The purpose of this study was to evaluate the effect of surface roughness (Ra) and finish of mechanically polished stainless steel (Ra = 0.26 +/- 0.05, 0.49 +/- 0.10, and 0.69 +/- 0.05 microm) and electropolished stainless steel (Ra = 0.16 +/- 0.06, 0.40 +/- 0.003, and 0.67 +/- 0.02 microm) on Listeria adhesion and biofilm formation. A four-strain cocktail of Listeria monocytogenes was used. Each strain (0.1%) was added to 200 ml of tryptic soy broth (TSB), and coupons were inserted to the mixture for 5 min. For biofilm formation, coupons with adhesive cells were incubated in 1:20 diluted TSB at 32 degrees C for 48 h. The experiment was performed by a randomized block design. Our results show that the level of Listeria present after 48 h of incubation (mean = 7 log CFU/cm2) was significantly higher than after 5 min (mean = 6.0 log CFU/cm2) (P < 0.01). No differences in initial adhesion were seen in mechanically finished (mean = 6.7 log CFU/cm2) when compared with electropolished stainless steel (mean = 6.7 log CFU/cm2) (P > 0.05). Listeria initial adhesion (values ranged from 5.9 to 6.1 log CFU/cm2) or biofilm formation (values ranged from 6.9 to 7.2 log CFU/cm2) was not significantly correlated with Ra values (P > 0.05). Image analysis with an atomic force microscope showed that bacteria did not colonize the complete surface after 48 h but were individual cells or grouped in microcolonies that ranged from 5 to 10 microm in diameter and one to three cell layers in thickness. Exopolymeric substances were observed to be associated with the colonies. According to our results, electropolishing stainless steel does not pose a significant advantage for food sanitation over mechanically finished stainless steel.     

Strain-specific differences in the attachment of Listeria monocytogenes to alfalfa sprouts

Contamination of fresh produce with Listeria monocytogenes has resulted in outbreaks of systemic listeriosis and febrile gastroenteritis. Recalls of alfalfa sprouts have occurred due to contamination with L. monocytogenes. Alfalfa sprouts were used as a preharvest model to study the interaction with this human pathogen. Seventeen strains were assessed for their capacity to colonize alfalfa sprouts, and strain-specific differences (not related to source, serotype, or lineage) were revealed when the sprout irrigation water was changed daily. Two of the strains colonized and attached to the sprouts very well, reaching levels of more than 5 log CFU per sprout. The remaining strains varied in their final levels on sprouts between less than 1 to 4.7 log CFU per sprout. All of the L. monocytogenes strains grew to equivalent levels on the sprouts when the irrigation water was not changed, suggesting the differences observed with regular changing of the water resulted from differences in attachment. Further analysis of the best colonizing strains indicated that only between 0.3 and 1 log CFU per sprout could be removed by additional washing of the sprout, and the presence of normal sprout bacteria did not compete with the L. monocytogenes strains on the sprouts. The poorest colonizing strain was able to grow in the irrigation water during the experiment but could not attach to the sprouts. Microscopic examination of the sprouts with L. monocytogenes expressing the green fluorescent protein indicated that L. monocytogenes was associated with the root hairs of the sprouting alfalfa, with few to no cells visible elsewhere on the sprout.     

Persistent Listeria monocytogenes strains show enhanced adherence to food contact surface after short contact times

Adherence of 3 persistent and 14 nonpersistent Listeria monocytogenes strains to stainless steel surfaces after short and long contact times was investigated. L. monocytogenes strains were obtained from poultry plants and an ice cream plant throughout several years. Adherence tests were performed in tryptic soy broth at 25 degrees C for 1, 2, and 72 h. Test surfaces were rinsed after the contact time, and attached cells were stained with acridine orange and enumerated with an epifluorescence microscope. The persistent poultry plant strains showed adherence 2- to 11-fold higher than the nonpersistent strains following 1- and 2-h contact times. The adherence of the persistent ice cream plant strain after 1- and 2-h contact times was higher than most of the nonpersistent strains. Seven of 12 nonpersistent ice cream strains showed an adherence of less than half that of the persistent strain. After 72 h, the differences in adherence were not as marked, since half the nonpersistent strains had reached adherence levels comparable with the persistent strains. In fact, three nonpersistent strains showed even higher adherence than the persistent strains. Thus, results of this study reveal that persistent L. monocytogenes strains show enhanced adherence at short contact times, promoting their survival in food processing facilities and possibly having an effect on initiation of persistent plant contamination.     

Comparison of the cell surface properties and growth characteristics of Listeria monocytogenes and Listeria innocua

Growth kinetics and physicochemical surface properties were compared for three Listeria strains with differing degrees of virulence: L. monocytogenes LO28; its isogenic, nonhemolytic mutant L. monocytogenes Bof415; and a nonvirulent species, L. innocua (strain Lin9). The influences of growth stage (mid-exponential phase, early stationary phase, and mid-stationary phase) and culture temperature (20 and 37 degrees C) were assessed by determining the electrical properties and the hydrophobic-hydrophilic and Lewis acid-base characteristics of the three strains. L. innocua, although taxonomically very similar to L. monocytogenes, exhibited physicochemical surface properties that differed significantly from those of L. monocytogenes LO28 and L. monocytogenes Bof415. Indeed, under our experimental conditions, L. innocua cells presented a more marked electronegative character (particularly when cultured at 20 degrees C), as well as greater variability in their Lewis acid-base characteristics as a function of temperature and growth stage. Furthermore, the growth kinetics of the three strains revealed the onset of a decay phase after 16 h of culture at 37 degrees C for the L monocytogenes Bof415 mutant. All of these results demonstrate that under our experimental conditions, the growth and/or physicochemical characteristics of the slightly pathogenic or nonpathogenic Listeria strains (Bof415 and Lin9) differed from those of the virulent strain (L. monocytogenes LO28). Consequently, the use of Listeria strains recognized as nonvirulent appeared to provide a model that was not fully suitable for simulating the bioadhesive behavior of the pathogenic strains involved in foodborne diseases.     

Assessing biofilm formation by Listeria monocytogenes strains

When a microtitre plate assay was used to quantify biofilm production by Listeria monocytogenes strains following growth in Tryptone Soy Broth (TSB) for 48 h at 20 degrees C, 127 of 138 strains (92.0%) were classified as weak, 9 of 138 strains (6.5%) as moderate and only 2 of 138 strains (1.5%) as strong biofilm formers. The strains included environmental, animal, food (persistent and sporadic strains) and clinical isolates previously typed using esterase electrophoresis (ESE) and multi-locus enzyme electrophoresis (MEE). Strains from different sources produced similar quantities of biofilm, whereas biofilm production by ESE type II strains, irrespective of source, was greater than that observed for other ESE types. No correlation between MEE type and biofilm production was observed. A Petri dish assay which allowed parallel quantification and microscopic examination of biofilms was used to examine biofilm formation by selected L. monocytogenes strains during growth in TSB for 14 days at 20 degrees C. Results from these assays showed that following prolonged incubation, some L. monocytogenes strains categorized as weak biofilm formers by the 48 h microtitre assay, were able to form biofilms similar in terms of quantity and structure to those produced by strains classified as strong or medium biofilm formers. Results from 14-day Petri dish assays confirmed 48 h microtitre assays regarding greater biofilm production by ESE type II strains compared to other ESE types of L. monocytogenes. Biofilm production was similar for ESE type II persistent and sporadic food isolates but reduced for ESE type II clinical strains.     

Effect of temperature and growth media on the attachment of Listeria monocytogenes to stainless steel

Listeria monocytogenes ATCC 19111 cultivated in nutrient-rich medium (brain heart infusion, BHI) or starved in minimal medium (10% filter sterilized pond water and 90% sterilized distilled water) were investigated for their initial attachment to austenitic stainless steel No. 4 with satin finish at 4 °C, 20 °C, 30 °C, 37 °C, or 42 °C. A droplet (10 μl) containing  10⁷ CFU/ml of L. monocytogenes suspended in BHI or minimal medium was placed on the stainless steel surface. After holding in saturated humidity for 3 h at the desired temperature the surface was washed and prepared for scanning electron microscopy (SEM). Using SEM, attachment of L. monocytogenes was determined by counting cells remaining on the surface. When L. monocytogenes cultivated in BHI were used, with the exception of the number of attached cells being lower at 42 °C than at 37 °C and 30 °C, the number of attached cells increased with increasing temperature (P < 0.05). When L. monocytogenes starved in minimal medium were used, the number of attached cells also increased with increasing attachment temperature (P < 0.05), but the number of attached cells at 42 °C was lower than that at the other temperatures. The attachment of L. monocytogenes to stainless steel surface was greater when cultivated in rich medium of BHI vs starved in the minimal medium.     

Elimination of Listeria monocytogenes on hotdogs by infrared surface treatment

ABSTRACT:  The objective of this research was to develop an infrared pasteurization process with automatic temperature control for inactivation of surface-contaminated Listeria monocytogenes on ready-to-eat meats such as hotdogs. The pasteurization system contained 4 basic elements: an infrared emitter, a hotdog roller, an infrared sensor, and a temperature controller. The infrared sensor was used to monitor the surface temperature of hotdogs while the infrared emitter, modulated by a power controller, was used as a heating source. The surface temperature of hotdogs was increased to set points (70, 75, 80, or 85 °C), and maintained for bacterial kill. The infrared surface pasteurization was evaluated using hotdogs that were surface-inoculated with a 4-strain L. monocytogenes cocktail to an average initial inoculum of 7.32 log (CFU/g). On the average 1.0, 2.1, 3.0, or 5.3 log-reduction in L . monocytogenes was observed after the surface temperature of hotdogs was increased to 70, 75, 80, or 85 °C, respectively. Holding the sample temperature led to additional bacterial inactivation. With a 3 min holding at 80 °C or 2 min at 85 °C, a total of 6.4 or 6.7 logs of L. monocytogenes were inactivated. This study demonstrated that the infrared surface pasteurization was effective in inactivating L. monocytogenes in RTE meats.     

Adaptive and cross-adaptive responses of persistent and non-persistent Listeria monocytogenes strains to disinfectants

Persistent and non-persistent Listeria monocytogenes strains were tested for initial resistance and adaptive and cross-adaptive responses towards two quaternary ammonium compounds, alkyl-benzyl-dimethyl ammonium chloride and n-alkyldimethyl ethylbenzyl ammonium chloride, one tertiary alkylamine, 1,3-propanediamine-N-(3-aminopropyl)N-dodecyl, sodium hypochlorite and potassium persulphate. The initial resistance of two persistent and two non-persistent L. monocytogenes strains was observed to differ. Both types of strains adapted after a 2-h sublethal exposure to the quaternary ammonium compounds and the tertiary alkylamine, the highest increase in the minimum inhibitory concentration (MIC) being 3-fold. Progressively increasing disinfecting concentrations at 10 and 37 degrees C resulted in adaptation of L. monocytogenes to all disinfectants except potassium sulphate. The highest observed increase in MIC was over 15-fold, from 0.63 to 10 microg/ml of n-alkyldimethyl ethylbenzyl ammonium chloride. All strains reached approximately similar MICs. Stability of the increased resistance was tested by measuring MICs every seventh day for 28 days. The increased resistance to sodium hypochlorite disappeared in 1 week, but the quaternary ammonium compounds and the tertiary alkylamine showed increased resistance for 28 days. These results suggest that cellular changes due to adaptive responses continue to have an effect on the resistance some time after the exposure. All disinfectants were shown to cause cross-adaptation of L. monocytogenes, the highest increase in MIC being almost 8-fold. The only agent that L. monocytogenes could not be shown to cross-adapt to was potassium persulphate which did, however, cause cross-adaptation to the other disinfectants. The mechanism behind these adaptive responses seemed to be non-specific as cross-adaptation was observed not only between related but also unrelated disinfectants. These findings suggest that sustaining high disinfectant effectiveness may be unsuccessful by rotation, even when using agents with different mechanisms of action.     

DNA fingerprinting of Listeria monocytogenes strains by pulsed-field gel electrophoresis

Chromosomal DNA of Listeria monocytogenes strains was digested with SmaI restriction enzyme, and the resulting fragments were separated by pulsed-field gel electrophoresis. Distinctive banding patterns were obtained from the strains of major foodborne disease serotypes 1/2a, 1/2b and 4b. Matched sets of clinical and food isolates revealed close similarity among strains from a given episode and distinct differences among strains from separate episodes.     

Dairy farm reservoir of Listeria monocytogenes sporadic and epidemic strains

Identifying the reservoirs of a pathogen is vital for control of sporadic disease and epidemics. Listeria monocytogenes is a zoonotic foodborne pathogen that is responsible for 28% of food-related deaths in the United States annually, as well as a major cause of massive product recalls worldwide. To examine the role of the dairy farm as a potential source or reservoir for L. monocytogenes subtypes shown to cause human listeriosis, we compared the pulsed-field gel electrophoresis (PFGE) restriction enzyme digestion profiles of L. monocytogenes dairy farm-associated strains (milk, environmental, and bovine) to human sporadic and epidemic disease strains. Twenty-three percent of human sporadic strains had PFGE patterns identical to that of farm isolate(s). Additionally, three farm environmental strains and one human sporadic strain had a PFGE pattern identical to a strain of L. monocytogenes responsible for the 1985 California epidemic. These data indicate that this epidemic strain continues to cause sporadic human illness and has a potential dairy farm as a reservoir.     

Effects of storage atmosphere on Listeria monocytogenes and competing microflora using a surface model system

A solid-surface model system was used to study the effects of gas atmospheres encountered in modified atmosphere packaging of vegetables on the survival and growth of Listeria monocytogenes and competing micro-organisms. The effects of increasing CO₂ levels (from 5% to 20%), 100% N₂ and 3% O₂ were determined. The model system allowed for estimation of the growth of L. monocytogenes alone or in the presence of competing microflora. CO₂ concentrations of 5–10% (with 5% O₂ in N₂) had no inhibitory effect, by comparison with air, on the growth and survival of pure cultures of L. monocytogenes . At 20% CO₂ population densities were reduced up to day 8, but the final population densities reached were not. An atmosphere of 100% N₂ allowed survival of pure cultures of L. monocytogenes , but populations did not significantly change ( P  > 0.05) during storage, whereas a low O₂ (3%, balance N2) atmosphere allowed significant growth ( P  < 0.05) of L. monocytogenes . Growth and inhibitory activities of Enterobacter cloacae and E. agglomerans were inversely related to the concentration of CO₂. By contrast, the growth and anti-listerial activities of Leuconostoc citreum increased with elevated CO₂ concentrations. In the low O₂ atmosphere, L. monocytogenes grew considerably better in the presence of populations from the indigenous microflora of lettuce than when in pure culture. The results indicate that the gas atmospheres present within modified atmosphere packages of minimally processed vegetables may affect the interactions between the pathogen and the natural competitive microflora sufficiently to indirectly enhance L. monocytogenes growth.     

Growth potential of Listeria monocytogenes strains in mixed ready-to-eat salads

In this study, a microbiological challenge test in three artificially contaminated retail mixed mayonnaise-based ready-to-eat salads stored at refrigerator temperatures (3 °C and 7 °C) for 48 h was carried out. Shrimp-tomato salad, smoked ham salad and garlic cheese salad were separately contaminated by a suspension of particular Listeria monocytogenes strains. The number of L. monocytogenes, Enterobacteriaceae, staphylococci and total plate count (CFU/g) was determined. Listeria monocytogenes growth potential in the salads was calculated and evaluated.A significant increase in total plate count and L. monocytogenes count throughout storage of all three investigated salads was found. Enterobacteriaceae levels were high at the beginning in all salads but significantly (p < 0.05) decreased throughout the experiment depending on the temperature.All investigated L. monocytogenes strains demonstrated growth at both temperatures but expressed different growth potential. Especially garlic cheese salad and smoked ham salad were able to support the growth of Listeria. Shrimp-tomato salad supported growth the least. The growth potential increased with the increasing temperature and exceeded 0.5 log₁₀ CFU/g in many cases. If the potential for growth is > 0.5 log₁₀ CFU/g, food products can potentially endanger human health. Reference strain (ATCC 7644) showed the least growth potential almost in all cases in comparison with strains isolated from frozen pollock loins and from thermally treated specialty sausage containing preservatives. To eliminate the occurrence of microbiological risks, the shelf-life of the studied salads was estimated.     

Survival of Listeria monocytogenes strains in a dry sausage model

The survival of five inoculated Listeria monocytogenes strains (DCS 31, DCS 184, AT3E, HT4E, and HR5E) was studied in dry fermented sausages prepared using two different starter cultures (starter A and B) with or without a protective Lactobacillus plantarum DDEN 2205 strain. L. monocytogenes was detected throughout ripening in every sausage sample in which the L. plantarum DDEN 2205 strain had not been used. The use of either starter A, with a high concentration of protective culture, or starter B, with a low concentration of protective culture, resulted in L. monocytogenes-negative sausages after 17 days of ripening. Differential survival was noted among the L. monocytogenes strains during fermentation. Strains AT3E and DCS 31 survived in sausages with protective cultures more often than did the other strains, whereas HT4E and HR5E were inhibited during ripening by all starter and protective cultures used. Protective cultures such as L. plantarum may be used as part of a hurdle strategy in dry sausage processing, but variations in susceptibility of different L. monocytogenes strains can create problems if other hurdles are not included.     

基于全基因组测序的单核细胞增生李斯特菌食品分离株分子特征分析

目的 了解肇庆市单核细胞增生李斯特菌(以下简称单增李斯特菌)食品分离株的基因组特征、毒力岛的携带情况及遗传多样性。方法 对肇庆市13株单增李斯特菌食品分离株进行全基因组测序,将组装好的contings/Scaffolds上传至在线分析平台Center for Genomic Epidemiology、Rast和VFanalyzer进行基因组注释与毒力因子基因鉴定,并应用基于全基因组测序的单核苷酸多态性分型(wg-SNPs)方法与美国生物技术信息中心(NCBI)上获取的25株国内外单增李斯特菌的基因组进行遗传进化分析。结果 13株单增李斯特菌食品分离株的基因组大小为2.82～3.04 Mb,CG含量为37.9%～38.1%,可分为6个ST型(ST1、ST3、ST8、ST59、ST87、ST101),分属于6个克隆复合群(CC1、CC3、CC8、CC59、CC87、CC101)。其中,ST3型菌株均携带LIPI-3毒力岛基因,ST87型菌株均携带完整LIPI-4毒力岛基因。wg-SNPs遗传进化分析显示13株单增李斯特菌食品分离株可分为2个进化分支,其中ST3型菌株位于进化树的根部,与其他ST型菌株进化上存在差异。结论 肇庆市单增李斯特菌食品分离株以高毒力的ST87型和ST3型为流行型,并发现一株同时携带LIPI-1～LIPI-4毒力岛的ST87型菌株,应加强对此类菌株的监测,警惕高毒力菌株在肇庆市引起感染性暴发的风险。     

Bacteriocin (monocin) interactions among Listeria monocytogenes strains.

Monocin interactions of 97 strains of Listeria monocytogenes were assessed using an improved production method and standardisation of the monocins against the type strain of L. ivanovii. Monocins were resistant to trypsin, sensitive to heating at 56 degrees C for 30 min and stable at 4 degrees C. Only serovar 4 strains acted as indicators. A typing system using 8 producer and 11 indicator strains showed poor discrimination.     

食品中单核细胞增生李斯特菌株的分离及聚合酶链式反应鉴定

目的 建立单核细胞增生李斯特菌的聚合酶链式反应(PCR)检测方法,了解市售食品中单核细胞增生李斯特菌的污染情况.方法 采集成都市市售生畜肉、生禽肉、熟肉制品、水产品、生食蔬菜以及其他熟食等食品样品共135份,采用李氏增菌肉汤(LB1,LB2)进行初增菌,应用选择性分离培养基PALCAM和在TSA-YE平板上进行分离,利用单增李斯特显色平板进行鉴定;根据李斯特菌的特异性基因iap基因设计引物,采用PCR方法检测所有分离的李斯特菌株;根据单增李斯特菌的特异性基因hly基因和prfA基因设计引物检测单核细胞增生李斯特菌株.结果 135份样品中共检出李斯特菌17株,检出率为12.6％;其中单核细胞增生李斯特菌4株,检出率为3.0％.结论 本研究建立的PCR方法具有特异性,本市市售食品不同程度受到李斯特菌的污染.