Performance testing of six chromogenic ALOA-type media for the detection of Listeria monocytogenes

Aims:  The aim of the study was to test the performance of commercially available chromogenic plating media for detection and enumeration of the food-borne pathogen Listeria monocytogenes . A wide range of chromogenic media similar to Agar Listeria according to Ottaviani and Agosti (ALOA) were compared using PALCAM agar, according to van Netten et al.
Methods and Results:  Six chromogenic media similar to ALOA were challenged for inclusivity and exclusivity. Additionally, the ability of chromogenic agars to facilitate growth of stressed L. monocytogenes strains and mixed cultures with competitive non-Listeria strains was estimated. Finally, we tested the detection and enumeration of L. monocytogenes in artificially inoculated and naturally contaminated food samples. The results of this study indicated that chromogenic media are a good supplementation to PALCAM agar. A single application is not advisable, as the specificity of chromogenic agars is frequently insufficient (50·0–88·9%), particularly in food samples with a complex microflora.
Conclusions:  The competitive flora of food samples is able to overgrow low numbers of L. monocytogenes , especially in half-Fraser enrichment. This might lead to the underestimation of L.   monocytogenes positive samples.
Significance and Impact of the Study:  Although many evaluation studies of chromogenic agar have been published recently, harmonized validation strategies are lacking. This survey provides a new concept for stepwise testing of plating media.     

An improved selective isolation medium for the recovery of Listeria monocytogenes from smoked fish

AIMS: The aim of this study was to improve the selective isolation of Listeria monocytogenes from smoked haddock fillets. METHODS AND RESULTS: Listeria selective agar (LSA)--Oxford formulation was supplemented with 25 microg x ml(-1) of colistin sulphate and 30 microg x ml(-1) of nalidixic acid. Inocula from four smoked haddock fillets produced colonies (approx. 2-13 bacteria x g(-1)), identified as L. monocytogenes, on LSA supplemented with antimicrobial compounds (MLSA). Moreover, there was only negligible evidence of bacteria which were not L. monocytogenes on MLSA. In contrast, LSA supported dense bacterial growth, which was not equated with L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The modified medium permitted the recovery of L. monocytogenes from smoked haddock fillets and reduced the growth of contaminating bacteria.     

免疫磁珠富集技术联合选择性培养基快速检测单增李斯特菌

单增李斯特菌是一种危害极大的食源性致病菌,建立快速及特异的检测方法对于食品安全监控尤为重要。文中联合免疫磁珠与选择性培养基对不同浓度(101~105CFU/mL)单增李斯特菌进行检测,并对3种李斯特菌、金黄色葡萄球菌及副溶血弧菌进行交叉试验;同时模拟食物污染,探索免疫磁珠-平板法检测样品的检测限以及该方法的最快检测时间。结果显示特异性免疫磁珠联合选择性平板法可检出浓度为103CFU/mL及以上的单增李斯特菌;牛奶样品仅需6 h增菌能被检出,检测限为0.7 CFU/mL。联合使用免疫磁珠富集技术与选择性培养基,能在30 h内完成对牛奶样品的检测,较国标法减少38 h以上,且具有同等的灵敏度。     

Investigation of relationships between marine diatoms and the bacterium Listeria monocytogenes

Relationships between marine diatoms and the bacterium Listeria monocytogenes have been studied by routine algological methods and high-resolution video-enhanced differential interference contrast light microscopy. The study showed that the relationship between the listeria and the benthic diatom Navicula sp. has a parasitic character, whereas the relationship between the listeria and the planktonic diatom Phaeodactylum tricornutum is protocooperative.     

一株抑制单增李斯特菌感染的S.epidermidis G26菌株的分离筛选

本研究从BALB/c小鼠粪便中分离筛选得到一株具有抑制单增李斯特菌(Listeria monocytogenes,Lm)感染的G26菌株。通过16S rDNA鉴定分离株G26为表皮葡萄球菌(Staphylococcus epidermidis G26)。体外试验证明G26能够显著抑制Lm生长;动物实验表明,分离株G26能够降低单增李斯特菌感染小鼠的死亡率,减少单增李斯特菌在小鼠肠道内的含量和粪便检出量。结果表明,分离株G26可以作为一种具有治疗单增李斯特菌感染作用的潜在的益生菌。     

Evaluation of a new chromogenic agar for the detection of Listeria in food

AIM: Rapid identification of Listeria in food is important in protecting consumers from infection. The development of chromogenic media such as agar Listeria according to Ottaviani and Agosti (ALOA) has allowed more rapid detection of Listeria monocytogenes, with presumptive identification of this pathogenic species after only 24 h of incubation. The aim of this study was to evaluate Oxoid chromogenic Listeria agar (OCLA) in comparison with ALOA and a traditional, nonchromogenic medium, Oxford agar. METHODS AND RESULTS: Media were compared using pure cultures, spiked food samples and naturally contaminated samples. Whilst development of typical colony morphology took 48 h on Oxford agar, Listeria spp. were frequently detected after 24 h of incubation on OCLA and ALOA. There was no significant difference in recovery between the two chromogenic media. CONCLUSIONS: Results indicate that OCLA gives equivalent recovery of Listeria spp. compared with ALOA. Whilst L. monocytogenes was frequently detected after 24 h of incubation, a 48-h incubation time was necessary to ensure detection of both L. monocytogenes and other Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that a commercially available chromogenic medium other than ALOA is appropriate for use in the international standard method. The commercial availability of more than one medium will facilitate the more widespread use of the method, thus increasing confidence in the ability to detect L. monocytogenes in food in the presence of other Listeria spp.     

A new chromogenic medium for the isolation of Listeria spp

A new medium is described for the isolation of Listeria spp. from foods and environmental samples. It is based on a modified Oxford medium in which 3,4-cyclohexenoesculetin-beta-D-glucoside replaces aesculin. Positive colonies are intensely black with the advantage that the pigment does not diffuse into the medium. The medium, when tested alongside the US Department of Agriculture (spiked samples) and Food and Drug Administration (naturally contaminated samples) isolation procedures, performed significantly better than the current formulations (34% more confirmed positives from naturally contaminated samples) with a reduction of 1 d in the assay time for most samples.     

一种免疫生物传感器对单核细胞增生李斯特菌快速检测方法的初步研究

应用F0F1-ATP酶旋转分子马达和免疫技术相结合,建立免疫生物传感器快速检测技术。首先pH变化敏感荧光物质F1300标记到色素体(chrom atophore)的内表面,然后在F0F1-ATP酶上连接β亚基抗体-生物素-链亲和素-生物素-单核细胞增生李斯特菌多抗复合体,得到可以捕获单核细胞增生李斯特菌的免疫生物传感器。传感器上负载菌量不同,酶活性不同,酶活变化以pH敏感的荧光探针来感应,最后通过荧光扫描仪检测不同菌量负载下的荧光信号。结果表明,该方法对单核细胞增生李斯特菌标准菌株(ATCC 15313)的检测时间为4.5 h,检出浓度为100 CFU/孔。     

Evaluation of ALOA plating medium for its suitability to recover high pressure-injured Listeria monocytogenes from ground chicken meat

AIMS: To evaluate a chromogenic plating medium for the isolation of sublethally injured cells of Listeria monocytogenes from processed foods. METHODS AND RESULTS: The inactivation of L. monocytogenes at pressures up to 400 MPa and 12 degrees C in ground chicken meat was employed to examine the recovery of high-pressure injured cells. Before and after different repair incubation periods at 30 degrees C in a nonselective broth, samples were plated onto a selective and differential agar [Agar Listeria according to Ottaviani and Agosti (ALOA)] and in the same medium supplemented with 4% sodium chloride (ALOA-S), and incubated at 37 degrees C. Sublethally injured cells were able to grow when directly plated onto the ALOA medium, without a previous repair incubation period. However, only uninjured cells grew on the ALOA-S medium. CONCLUSIONS: Sublethally injured cells of L. monocytogenes can be quantified by subtracting counts on ALOA-S medium from counts on ALOA medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Possible applications include direct enumeration on ALOA of stressed cells of L. monocytogenes in foods with more than 100 colony forming units per gram.     

单核细胞增多性李斯特菌氨基肽酶Lmo1711体外表达及其酶活分析

对单核细胞增多性李斯特菌(简称单增李斯特菌)lmo1711基因编码的氨基肽酶进行克隆表达与纯化,并研究该重组蛋白的体外酶学特性。首先通过生物信息学分析预测Lmo1711与氨基肽酶家族成员的亲缘关系及关键活性位点的保守性。利用SWISS-MODEL模拟预测该蛋白的空间结构;构建Lmo1711原核表达载体并转化入E.coli Rosetta中,诱导表达重组目的蛋白,并利用镍离子亲和层析方法纯化目的蛋白;以氨基酸-对硝基苯胺偶联物为底物,Lmo1711通过水解底物N端氨基酸残基产生游离对硝基苯胺单体,405 nm处检测吸光值对该产物进行检测从而分析Lmo1711的酶学特性。在此基础上系统研究Lmo1711对不同氨基酸残基底物的催化特异性,及不同金属离子对该酶活性的影响。经原核表达纯化获得49.3 kDa的重组Lmo1711蛋白,与预测分子量一致;生物信息学分析推测Lmo1711属于M29氨基肽酶家族,且存在保守关键氨基酸活性位点(Glu250、Glu316、His345、Tyr352、His378、Asp380);酶活分析显示,Lmo1711具有较强的氨基肽酶活性,针对不同底物的结合和催化能力差异较大,对亮氨酸残基的亲和程度最高;Lmo1711氨基肽酶活性具有金属离子依赖性,Co~(2+)、Cd~(2+)、Zn~(2+)等多种金属离子均能显著增强其活性,其中Co~(2+)的激活效应最显著。本试验首次发现并证实,单增李斯特菌Lmo1711属于M29氨基肽酶家族成员,具有较强的催化活性,且对金属离子具有不同程度的依赖性。     

Virulence Phenotyping and Molecular Characterization of a Low-pathogenicity Isolate of Listeria monocytogenes from Cow's Milk

A low-pathogenicity isolate of Listeria monocytogenes from cow's milk,as screened in mouseand chicken embryonated egg models,was examined for virulence-related phenotypic traits.Correspondingvirulence genes (iap,prfA,plcA,hly,mpl,actA,plcB,InlA and InlB) were compared with L.monocytogenesreference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence.Al-though L.monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity,invitro growth and invasiveness and even had higher adhesiveness,faster intracellular growth and higherphospholipase activity in vitro,it was substantially less virulent than the strain 10403S in mouse and chickenembryo models (50% lethal dose:10~(8.14) vs.10~(5.49) and 10~(6.73) vs.10~(1.9),respectively).The genes prfA,plcA andmpl were homologous among L.monocytogenes strains H4,10403S and EGD (>98%).Genes iap,hly,plcB,InlA and InIB of L.monocytogenes 10403S had higher homology to those of strain EGD (>98%) than isolateH4.The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level,but 98.7% at the amino acid level.The actA gene of isolate H4 had deletions of 105 nucleotides correspondingto 35 amino acid deletions falling Within the proline-rich region.Taken together,this study presents someclues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletionmutations of actA.     

Virulence Phenotyping and Molecular Characterization of a Low-pathogenicity Isolate of Listeria monocytogenes from Cow＇s Milk

A low-pathogenicity isolate of Listeria monocytogenes from cow＇s milk, as screened in mouse and chicken embryonated egg models, was examined for virulence-related phenotypic traits. Corresponding virulence genes （iap, prfA, picA, hly, mpl, actA, plcB, InlA and lnlB） were compared with L. monocytogenes reference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence. Although L. monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity, in vitro growth and invasiveness and even had higher adhesiveness, faster intracellular growth and higher phospholipase activity in vitro, it was substantially less virulent than the strain 10403S in mouse and chicken embryo models （50% lethal dose： 10^8.14 VS. 10^5.49 and 10^6.73 VS. 10^1.9, respectively）. The genes prfA, picA and mpl were homologous among L. monocytogenes strains H4, 10403S and EGD （〉98%）. Genes iap, hly, plcB, lnlA and lnlB of L. monocytogenes 10403S had higher homology to those of strain EGD （〉98%） than isolate H4. The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level, but 98.7% at the amino acid level. The actA gene of isolate H4 had deletions of 105 nucleotides corresponding to 35 amino acid deletions falling within the proline-rich region. Taken together, this study presents some clues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletion mutations of actA.     

A novel real-time PCR-based method for the detection of Listeria monocytogenes in food

AIMS: A new real-time PCR-based method was developed for the detection of Listeria monocytogenes in food. METHODS AND RESULTS: A two-step enrichment involving a 24-h incubation in half-Fraser broth followed by a 6-h subculture in Fraser broth was used, followed by cell lysis and real-time PCR with primers and a TaqMan probe previously developed in our laboratory. When the method was evaluated with 144 naturally contaminated food samples, 44 were detected as positive by the PCR-based method and 42 by the standard method EN ISO 11290-1. With 61 food samples artificially contaminated at a level of 10(0) CFU per 25 g, 61 and 58 positive samples were detected by the respective methods. CONCLUSIONS: The developed real-time PCR-based method facilitated the detection of L. monocytogenes in food on the next day after the sample reception, with a reduction of false-positive results because of dead bacterial cells and false-negative results because of PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be used for L. monocytogenes detection in food as a faster alternative to current methods.     

Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: clues for evolution and pathogenicity

Listeria monocytogenes, an invasive opportunistic, food-borne pathogen, remains one of the leading causes of mortality from food-borne infections. The recently determined complete genome sequences of L. monocytogenes strain EGDe and of that of the closely related non-pathogenic species Listeria innocua strain CLIP11262 enhance our knowledge of the genetic basis of the virulence of L. monocytogenes and advance our understanding of the evolution of these Listeria species. Both genomes encode a high number of surface, transport and regulatory proteins. Comparison of the genome organisation revealed a perfect synteny between the two Listeria genomes. Comparison with other closely related bacteria also showed a high conservation in genome organisation among the Listeria, Staphylococcus and Bacillus group of low G+C content bacteria. Distinct G+C content of a number of strain-specific genes suggests intensive lateral gene transfer. The identification of a 55-kb locus encoding proteins with high homology to Salmonella enterica serovar Typhimurium vitamin B(12) synthesis proteins as well as those necessary for degradation of ethanolamine and propanediol further indicates acquisition of a complete metabolic pathway by horizontal gene transfer and a probable role of this locus in anaerobic growth in the host.     

单增李斯特菌lmo2192基因克隆与表达

[目的]对单增李斯特菌新疆绵羊脑炎临床分离株LM90SB2的lmo2192基因进行克隆及原核表达。[方法]利用PCR方法扩增lmo2192基因,连接p MD19-T载体进行克隆,筛选阳性菌进行测序比对。构建重组表达质粒p ET32a-2192,将其转入大肠杆菌感受态细胞,经诱导表达,利用SDS-PAGE与Western Blotting鉴定重组蛋白。[结果]扩增lmo2192基因序列长度为969 bp,与预期一致。该基因在大肠杆菌中大量表达,经SDS-PAGE和Western Blotting鉴定分析该产物为56 k Da左右的融合重组蛋白,与预期大小一致。[结论]成功克隆lmo2192基因,并获得大量表达,为进一步研究该基因功能奠定理论基础。     

Enhanced in vitro engulfment of Listeria monocytogenes by rabbit polymorphonuclear leukocytes in the presence of sera from immune rabbits

Abstract The role of immune serum in the engulfment of Listeria monocytogenes by polymorphonuclear leukocytes (PMNLs) of rabbits was documented employing an in vitro assay. Three serovarieties of L. monocytogenes , viz. serovars 1/2a, 4a and 4b, were separately incubated with PMNLs of nonimmune rabbit and high titre homologous or heterologous antisera. Normal rabbit serum was used as control. The number of L. monocytogenes per neutrophil was counted in stained smears in each assay and opsonic indices were calculated. Higher opsonic indices, i.e. 2.50, 2.09 and 2.56, for the three strains respectively, were obtained when bacteria were engulfed in the presence of homologous antisera as compared to when the bacterial cells were incubated with heterologous antisera, opsonic indices being in the range of 0.87 to 1.63. These results are indicative of a possible role of specific serum factors in at least the internalization of L. monocytogenes by PMNLs of rabbits and therefore in the host defenses against Listerial infections.     

大肠杆菌O157显色培养基检测效果的评价

【目的】评价显色培养基对大肠杆菌O157:H7(Escherichia coli O157:H7)的检测效果。【方法】本实验室研制的大肠杆菌O157显色培养基(HKM),与国外梅理埃、科玛嘉及国内厂家的同类产品及传统培养基CT-SMAC作比较,对相关菌株以及污染样品和实际样品进行对比测试。【结果】实验室研制的HKM大肠杆菌O157显色培养基与科玛嘉同类产品在特异性、灵敏度及检测效果方面均无明显差异,均优于梅里埃、国内厂家产品及CT-SMAC。【结论】HKM大肠杆菌O157显色培养基具有高检出率及高特异性的特点,具有较好的应用价值和前景。     

Molecular cloning and phylogenetic analysis of the small cytoplasmic RNA from Listeria monocytogenes

A molecular cloning strategy has been designed to isolate the gene that encodes the small cytoplasmic RNA (scRNA) component of bacterial signal recognition particles. Using this strategy a putative Listeria monocytogenes scRNA lambda gt11 recombinant clone was isolated. A previously described complementation assay developed to genetically select functional homologues of 4.5S RNA and scRNA of bacteria confirmed that the lambda gt11 recombinant clone isolated encoded for the scRNA from L. monocytogenes. A secondary structure for this scRNA is proposed and a phylogenetic comparison of the 276 base L. monocytogenes scRNA with previously characterised Gram-positive bacterial scRNAs is also presented.