Characterization of iap gene in Listeria monocytogenes strains isolated in Japan

Variation of the iap gene region (407bp) encoding an invasion-associated protein p60 was studied on 12 strains of Listeria monocytogenes of different origin in Japan. These 12 strains are known to have 2 types of serotype (1/2a and 4b) and have a diversity among the strains (Saito et al., 1998). The dye-primer cycle sequencing method was employed to determine the genomic structure, and the nucleotide sequences obtained were compared with those of reference strain SV 1/2a EGD. Differences found in the nucleotides were as follows; point mutations of 33 variations in 32 places; an insertion and 3 deletions of 3 bases; AAT position (po.) 1282-1283, and GCA po. 1307-1309, ACA po. 1412-1414, AAT po. 1439-1444, respectively. Different repeating numbers by 6 base unit, ACA AAT, were also found in the tandem repeat region (po. 1394-1423). Classification of 12 strains was attempted, then 8, 4 and 5 types were obtained from the point mutations, the insertions and deletions, and the repeating numbers, respectively. Consequently, 8 patterns were profiled regardless of each serotype. From these results, genomic structures were partially clarified in the iap gene 407bp of L. monocytogenes isolated in Japan. Then, the possibility of detailed epidemiology for L. monocytogenes infection using a combination of serotype and genome structure was suggested because of the previous polymorphism thought to be due to the nucleotide differences in the region.     

CO2- and Anaerobiosis-Induced Changes in Physiology and Gene Expression of Different Listeria monocytogenes Strains

Although carbon dioxide (CO₂) is known to inhibit growth of most bacteria, very little is known about the cellular response. The food-borne pathogen Listeria monocytogenes is characterized by its ability to grow in high CO₂ concentrations at refrigeration temperatures. We examined the listerial responses of different strains to growth in air, 100% N₂, and 100% CO₂. The CO₂-induced changes in membrane lipid fatty acid composition and expression of selected genes were strain dependent. The acid-tolerant L. monocytogenes LO28 responded in the same manner to CO₂ as to other anaerobic, slightly acidic environments (100% N₂, pH 5.7). An increase in the expression of the genes encoding glutamate decarboxylase (essential for survival in strong acid) as well as an increased amount of branched-chain fatty acids in the membrane was observed in both atmospheres. In contrast, the acid-sensitive L. monocytogenes strain EGD responded differently to CO₂ and N₂ at the same pH. In a separate experiment with L. monocytogenes 412, an increased isocitrate dehydrogenase activity level was observed for cells grown in CO₂-containing atmospheres. Together, our findings demonstrate that the CO₂-response is a partly strain-dependent complex mechanism. The possible links between the CO₂-dependent changes in isocitrate dehydrogenase activity, glutamate metabolism and branched fatty acid biosynthesis are discussed.     

Characteristics of nonpathogenic strains of Listeria monocytogenes

Five strains of nonpathogenic Listeria monocytogenes were characterized for (i) hemolysin production, (ii) cytolysis of Chinese hamster ovary (CHO) cells, and (iii) ability to attach and enter intestine 407 cells. Four of the five strains produced variable hemolysis and were weakly cytolytic for Chinese hamster ovary cells, whereas the other isolate was consistently hemolytic and strongly cytolytic for CHO cells. None of the strains was able to penetrate intestine 407 cells. In addition, two of the five strains were found to be nonmotile.     

Genetic transformation between strains of Listeria monocytogenes

Acid and alkaline phosphatase activities of microbial films colonizing glass surfaces were studied. Films developed in water with a high organic content were characterized by a high ratio of alkaline to acid phosphatase activity. Alkaline phosphatase activity of these films was enhanced by a period of prior heating at 60°C for 10 min. Microbial films developed in poorer water exhibited higher proportions of acid phosphatases and heat treatment had a less favourable effect on the alkaline phosphatase activity.     

Proteomic expression profiles of virulent and avirulent strains of Listeria monocytogenes isolated from macrophages

Listeria monocytogenes is able to survive and proliferate within macrophages. In the current study, the ability of three L. monocytogenes strains (serovar 1/2a strain EGDe, serovar 4b strain F2365, and serovar 4a strain HCC23) to proliferate in the murine macrophage cell line J774.1 was analyzed. We found that the avirulent strain HCC23 was able to initiate an infection but could not establish prolonged infection within the macrophages. By contrast, strains EGDe and F2365 proliferated within macrophages for at least 7 h. We further analyzed these strains by comparing their protein expression profiles at 0 h, 3 h, and 5 h post-infection using multidimensional protein identification technology coupled with electrospray ionization tandem mass spectrometry. Our results indicated that similar metabolic and cell wall associated proteins were expressed by all three strains at 3 h post-infection. However, increased expression of stress response and DNA repair proteins was associated with the ability to proliferate in macrophages at 5 h post-infection. By comparing the protein expression patterns of these three L. monocytogenes strains during intracellular growth in macrophages, we were able to detect biological differences that may determine the ability of L. monocytogenes to survive in macrophages.     

Pulse-electrotypes of Listeria monocytogenes strains, isolated in Moscow

The characterization of the pulse-electrotypes of L. monocytogenes, isolated in 2003-2004 in Moscow from different sources, is presented. Among the cultures, isolated from humans, one outbreak pulse electrotype was detected and from different objects in buildings where a wide variety of food products was produced several probably related and unrelated pulse-electrotypes were obtained. The conclusion was made that several independent L. monocytogenes clones existed on the territory of Moscow, and many products supplied to retail trade and public catering enterprises were contaminated with these clones. Pulse electrophoresis was shown to be the most effective method for intraspecific typing and the study of the molecular epidemiology of listeriosis. Grounds for the necessity to improve the microbiological diagnostics of L. monocytogenes infection are given.     

Variation in biofilm formation among strains of Listeria monocytogenes

Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells may produce multicellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between biofilm formation and disease incidence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in biofilm formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased biofilm formation relative to nonpersistent strains. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high-biofilm-producing strains. Scanning electron microscopy showed that a high-biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-biofilm-forming strain produced a thin, patchy biofilm. These data are consistent with data on persistent strains forming biofilms but do not support a consistent relationship between enhanced biofilm formation and disease incidence.     

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。     

单核细胞增生性李斯特菌prfA基因缺失株的构建及其生物学特性

【目的】单核细胞增生性李斯特菌(Lm)是人兽共患李斯特菌病的病原菌,其致病性与调控因子PrfA蛋白作用下毒力基因的表达有着密切关系,本文初步探讨了PrfA蛋白对细菌毒力因子的调控作用。【方法】利用同源重组技术对血清型分别为1/2a和4b的LM4、F4636进行prfA基因的敲除,并构建其回复突变株,对获得的突变株LM4ΔprfA、F4636ΔprfA进行生物学特性研究。【结果】实验结果表明:两株缺失株的溶血活性丧失、回复突变株的溶血活性得到恢复,突变株还丧失磷脂酶活性,黏附和侵袭特性显著下降(P<0.05),对BALB/c小鼠的半数致死剂量提高了105个数量级。【结论】由此表明,PrfA蛋白对hly、plcB、inl家族基因的表达及细菌毒力具有重要的调控作用。prfA基因缺失株的构建为进一步研究PrfA蛋白的调控功能提供了材料,为研究其在Lm致病性中的作用奠定了基础。     

Determination of virulence of different strains of Listeria monocytogenes and Listeria innocua by oral inoculation of pregnant mice.

A pregnant mouse model was developed to follow the course of infection after peroral inoculation with six different strains of Listeria monocytogenes and one strain of Listeria innocua. Tissues were sampled and analyzed by microbiologic and histologic methods for 5 days postinoculation. In gnotobiotic pregnant BALB/c mice, L. monocytogenes Scott A (SA), serotype 4b, colonized the gastrointestinal tract, translocated to the livers and spleens of mice by day 1 postinoculation, and multiplied in these tissues until day 4. Infection of the placental tissues occurred by days 3 and 4 and was followed by infection of the fetuses. Little damage of colonic and cecal tissues was evident by histologic examination. Livers and spleens showed a cellular immune response; a similar immune response was not detected in the placentas or fetuses. A rough variant of L. monocytogenes SA which was as virulent as the parent strain in mice when injected intraperitoneally was less virulent perorally and did not consistently infect the fetuses. L. monocytogenes ATCC 19113, serotype 3a, did not colonize the gastrointestinal tract, nor was it isolated from any internal tissue. L. monocytogenes strains of serotypes 1/2a and 1/2b behaved like the SA strain in this mouse model. L. innocua colonized the gastrointestinal tract and translocated to the livers and spleens but did not survive in these organs and rapidly disappeared without infecting placental and fetal tissues. In comparison with gnotobiotic mice, conventional pregnant mice inoculated with L. monocytogenes strains showed less consistent infection. These results suggest that the gnotobiotic pregnant mouse is a useful model for detecting differences in virulence relating to colonization, invasiveness, and uteroplacental infection which cannot be detected by intraperitoneal inoculation of mice.     

Determination of virulence of different strains of Listeria monocytogenes and Listeria innocua by oral inoculation of pregnant mice.

A pregnant mouse model was developed to follow the course of infection after peroral inoculation with six different strains of Listeria monocytogenes and one strain of Listeria innocua. Tissues were sampled and analyzed by microbiologic and histologic methods for 5 days postinoculation. In gnotobiotic pregnant BALB/c mice, L. monocytogenes Scott A (SA), serotype 4b, colonized the gastrointestinal tract, translocated to the livers and spleens of mice by day 1 postinoculation, and multiplied in these tissues until day 4. Infection of the placental tissues occurred by days 3 and 4 and was followed by infection of the fetuses. Little damage of colonic and cecal tissues was evident by histologic examination. Livers and spleens showed a cellular immune response; a similar immune response was not detected in the placentas or fetuses. A rough variant of L. monocytogenes SA which was as virulent as the parent strain in mice when injected intraperitoneally was less virulent perorally and did not consistently infect the fetuses. L. monocytogenes ATCC 19113, serotype 3a, did not colonize the gastrointestinal tract, nor was it isolated from any internal tissue. L. monocytogenes strains of serotypes 1/2a and 1/2b behaved like the SA strain in this mouse model. L. innocua colonized the gastrointestinal tract and translocated to the livers and spleens but did not survive in these organs and rapidly disappeared without infecting placental and fetal tissues. In comparison with gnotobiotic mice, conventional pregnant mice inoculated with L. monocytogenes strains showed less consistent infection. These results suggest that the gnotobiotic pregnant mouse is a useful model for detecting differences in virulence relating to colonization, invasiveness, and uteroplacental infection which cannot be detected by intraperitoneal inoculation of mice.     

Atypical Listeria monocytogenes serotype 4b strains harboring a lineage II-specific gene cassette

Listeria monocytogenes is the etiological agent of listeriosis, a severe food-borne illness. The population of L. monocytogenes is divided into four lineages (I to IV), and serotype 4b in lineage I has been involved in numerous outbreaks. Several serotype 4b epidemic-associated clonal groups (ECI, -II, and -Ia) have been identified. In this study, we characterized a panel of strains of serotype 4b that produced atypical results with a serotype-specific multiplex PCR and possessed the lmo0734 to lmo0739 gene cassette that had been thought to be specific to lineage II. The cassette was harbored in a genomically syntenic locus in these isolates and in lineage II strains. Three distinct clonal groups (groups 1 to 3) were identified among these isolates based on single-nucleotide polymorphism-based multilocus genotyping (MLGT) and DNA hybridization data. Groups 1 and 2 had MLGT haplotypes previously encountered among clinical isolates and were composed of clinical isolates from multiple states in the United States. In contrast, group 3 consisted of clinical and environmental isolates solely from North Carolina and exhibited a novel haplotype. In addition, all group 3 isolates had DNA that was resistant to MboI, suggesting methylation of adenines at GATC sites. Sequence analysis of the lmo0734 to lmo0739 gene cassette from two strains (group 1 and group 3) revealed that the genes were highly conserved (>99% identity). The data suggest relatively recent horizontal gene transfer from lineage II L. monocytogenes into L. monocytogenes serotype 4b and subsequent dissemination among at least three distinct clonal groups of L. monocytogenes serotype 4b, one of which exhibits restrictions in regional distribution.     

Identification and characterization of an essential gene in Listeria monocytogenes using an inducible gene expression system

The Listeria monocytogenes gene lmo1594 is a homolog of the Bacillus subtilis cell division gene ezrA. EzrA is a negative regulator of FtsZ ring formation, which is required for efficient cell division as it regulates the frequency and position of Z-rings in the cell and prevents aberrant polar cell division. Previously identified as a putative high pressure (HP) resistance mechanism; conferring enhanced barotolerance when heterologously expressed against an Escherichia coli background; the aim of the current study was to investigate whether lmo1594 plays a role in listerial barotolerance. When the creation of a deletion mutant proved unsuccessful, the role of lmo1594 was addressed by creating a conditional knockout mutant which demonstrated that the gene is in fact essential for cell survival and growth in L. monocytogenes. In order to investigate the effect of lmo1594 on barotolerance, the gene was over-expressed. The over-expression of lmo1594 increased survival levels in L. monocytogenes treated at 300 MPa, but survival levels similar to those of the wild-type strain were observed when treated at a higher pressure (≥400 MPa). In conclusion, this study reveals for the first time that lmo1594 is absolutely essential for listerial cell growth and survival, and also plays an important role in listerial barotolerance.     

Swift and definite serotyping for isolated Listeria monocytogenes strains

The serotype is most important for molecular epidemiological analysis of Listeria monocytogenes (L.m.) contaminating marketed meats. An improvement on the traditional method was thus attempted in the present study because of the requirement of swift and definite serotyping. In the determination of O-antigen, definite judgement was allowed by an immediate cooling at 80 degrees C after autoclaving the bacteria. In the determination of H-antigen, use of a culture plate without Craigie's tube yielded the active bacteria only by single culture. The stable and clear agglutination in many samples was also obtained with a microplate using less antiserum. The availability was confirmed with 123 strains and the serovar 1/2b was dominant in the Japanese strains.     

Adherence of Listeria monocytogenes strains to stainless steel coupons

An assay was developed to measure the number of Listeria monocytogenes cells adhering to stainless steel, and was used to investigate the adherence of 111 strains of the organism, which included representatives with respect to serotype, carriage of plasmids, source and persistence in the food processing environment. Growth and adherence curves of four L. monocytogenes strains over 48 h were obtained. While the growth curves of all four micro-organisms were seen to reach similar levels at stationary phase, there was still substantial variation among the adherence curves. In addition, a scatter-graph of growth vs adherence counts at 24 h showed poor correlation. These factors indicated that interstrain variation in adherence at stationary phase is due to factor(s) intrinsic to each strain of L. monocytogenes. Persistent strains were found to adhere in significantly greater numbers than sporadic strains, and variation was also found among serotypes, with serotype 1/2c showing significantly greater adherence than serotypes 1/2a and 4b; 4b strains were significantly higher than those of 1/2a strains. No significant difference was found between strains according to source or plasmid carriage.