The application of PCR fingerprinting to the differentiation of Yersinia enterocolitica strains isolated from humans and pigs

The distribution of different genotypes of Yersinia enterocolitica strains recovered from humans and from healthy pigs was investigated using PCR fingerprinting. The thirty six strains of Y. enterocolitica from humans, thirty five strains from pigs and Y. enterocolitica ATCC 9610 strain were included in this study. The tested strains of Y. enterocolitica belonged to O3 and O9 serogroups. The PCR fingerprinting using EAE5 primer (5' CTT AAT CTC AGT AAT GCT GGC CTT GG) made it possible to form five groups among the tested Y. enterocolitica strains. Two groups were very numerously represented by the tested strains. The thirty of Y. enterocolitica O3 strains from humans (thirty one of tested) and eighteen of Y. enterocolitica O3 strains from pigs (twenty of tested) belonged to one group. This group also included Y. enterocolitica ATCC9610 strain and four Y. enterocolitica O9 strains from pigs. All investigated Y. enterocolitica O9 strains from humans and the majority of Y. enterocolitica O9 strains isolated from pigs created a second, numerous group. The third genotype was created by two strains O9 from pigs, and the remaining two strains, isolated from pigs, belonging to O3 and O9 serogroups showed different binding patterns revealed by gel electrophoresis and created two other genotypes. The tested Y. enterocolitica strains which were isolated from humans formed only two groups but Y. enterocolitica strains isolated from pigs were found in five groups but such as the Y. enterocolitica strains from humans, the majority of strains from pigs were in first and second group. The Y. enterocolitica O3 strains regardless of their origin mostly represented the same PCR fingerprinting profile. The tested Y. enterocolitica O9 strains were more genetically diverse and represented four PCR fingerprinting profiles.     

铜绿假单胞菌对11种抗生素的耐药性差异

目的探讨不同标本分离铜绿假单胞菌对11种抗生素耐药性的差异,指导临床合理使用抗生素。方法按常规方法培养分离后,经VITEK-AMS60或VITEK-II自动微生物鉴定分析仪的鉴定,用Kirty-Bauer法做药物敏感试验。结果尿液与血液之间,以及尿液与伤口分泌物之间,分离株均对所检测的11种抗生素的耐药性差异有非常显著性（P〈0.01）,尿液与穿刺液分离菌对除亚胺培南和美洛培南外的9种抗生素的耐药性差异有非常显著性（P〈0.01）,血液与穿刺液、血液与伤口分泌物和穿刺液与伤口分泌物间,除穿刺液与伤口分泌物分离菌对美洛培南的耐药性差异有显著性（P〈0.05）外,其他差异均无显著性（P〉0.05）。结论不同标本来源铜绿假单胞菌对哌拉西林等11种抗生素存在耐药性差异。     

Characteristics of E.coli O157 strains isolated in Poland from clinical material and food samples

E. coli belonging to the O157 serological group are among the organisms isolated most frequently out of all the so called entero-hemorrhagic E. coli strains (EHEC). Since several years they have been isolated also in Poland. The purpose of the present study was determination on selected phenotypic and genotypic properties of E. coli O157 strains isolated in our country from clinical material samples and from food. The serotype of the strains was determined, together with the following properties regarded as pathogenicity markers of verotoxic E. coli strains such as absence of beta-glucuronidase activity and sorbitol fermentation ability, as well as production of verotoxins SLT I and/or SLT II and entero-hemolysin. Besides that, by the PCR method the fragments of the genes coding for verotoxins, intimin and enterohaemolysin were amplified. The products of PCR were analysed by the restriction enzyme analysis (RFLP). All verotoxic E. coli O157 strains isolated in Poland were analysed by the pulsed field gel electrophoresis of genomic DNA (PFGE). The studied group comprised E. coli O157 strains, among them 40 strains were isolated from human faeces and 5 from food. The remaining strains were the reference E. coli O157:H7 EDL 933 and G 5244 strains and strains from NIH collection. The obtained results showed that the tested strains were a very varying population. 21 of them (all isolated from food, 11 from faeces and 5 reference strains) belonged to serotype O157:H7, five were not peritrichous O157:NM and the remaining ones had other ciliary antigen than H7. All strains isolated from food, reference strains and only 3 O157:NM strains isolated from humans were verotoxic. The strains from food and two reference strains produced only SLT II, 2 of 3 strains isolated from humans and one reference strain also produced only SLT II and the other produced both verotoxins. Apart from these 13 verotoxic strains all remaining strains caused sorbitol fermentation.     

Differences in the initial events of infection of Botrytis cinerea strains isolated from tomato and grape

Various stages of the infection process among B. cinerea strains isolated from tomatoes or grapes, belonging to different genetic groups, were compared. It was found that strains of B. cinerea isolated from either grapes or tomatoes showed differences in adhesion patterns and in the percentage of germination on tomato cutin. In strains isolated from tomato the first stage of adhesion occurred faster than in strains isolated from grape. At the same time strains isolated from tomato showed a higher percentage of germination on tomato cutin than the other strains after 9 h of incubation. The production and isoenzymatic patterns of polygalacturonases, pectin methyl esterases, pectin lyases, p-nitrophenylbutyrate esterases and laccases by B. cinerea in solid-state fermentation also were analyzed. Correlation between the production of these enzymes and the origin of the strains was not found. On the other hand all strains produced different isoenzymes and a common pattern between the strains was not observed. The ability of B. cinerea strains to colonize tomato leaves also differs between the isolated strains obtained from grapes and tomato. Strains isolated from tomato were more virulent on tomato leaves than strains isolated from grapes.     

Rhizobium meliloti competitiveness and the alfalfa agglutinin

We have isolated two types of isolates having identical colony morphologies from stock cultures of two different Rhizobium meliloti strains. One isolate was agglutinated at a high-dilution titer (HA, highly agglutinable) of the alfalfa agglutinin and was sensitive to phage F20, and the other was agglutinated at a lower agglutinin titer (LA) and was sensitive to phage 16B. All LA isolates from the original slant produced nodules on alfalfa earlier than did HA strains from the original slant. When these HA and LA strains were mixed and used as the inoculum in both vermiculite and field soil in the laboratory, LA strains were always the predominant strains recovered from the nodules. LA strains were obtained from HA cells by selection for resistance to phage F20, and HA strains were obtained from LA cells by selection for resistance to phage 16B. All of the strains with the HA phenotype that were derived from LA strains by phage selection had the nodulation properties of the HA strains from the original slant. Two classes of strains with the LA phenotype were obtained from HA cells by phage selection. One was identical to the original LA strains from the slant, and the other had the nodulation properties of the HA strains. Thus, we have shown that some cell surface properties change the nodulation abilities of R. meliloti strains and, furthermore, that specific phages can be used to enrich for more competitive rhizobia.     

大连市两种来源的副溶血性弧菌分子分型

目的 分析食品来源、患者来源及2种来源的副溶血性弧菌之间的PFGE图谱的关系,从分子流行病学角度探讨2种来源的副溶血性弧菌的关联.方法 收集患者和食品2种来源的副溶血性弧菌178株,经限制性内切酶SfiI酶切,用脉冲场凝胶电泳方法进行电泳,凝胶成像仪获得电泳图谱,利用BioNumerics软件对图谱进行聚类分析.结果 ...     

Differentiation of Erwinia amylovora strains by pulsed-field gel electrophoresis.

Erwinia amylovora strains, isolated from several host plants in various geographic regions during different years, were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the DNA from lysed, agar-embedded cells with rare-cutting restriction enzymes. The banding patterns obtained with enzyme XbaI digests revealed significant differences among strains from different areas. North American strains E9 and Ea-Rb, a Rubus strain, were highly divergent from other E. amylovora strains. French strains were different from central European and English strains. E. amylovora strains from central Europe and New Zealand had identical PFGE patters, as had strains from Egypt, Greece, and Turkey. PFGE of genomic DNA from American and English strains gave rise to dissimilar patterns. Patterns of some American strains resembled those from strains isolated in other parts of the world. The restriction fragment length polymorphisms observed by PFGE analysis can be used to group strains and may give hints about the course of distribution of the plant disease. From the sizes of the restriction fragments obtained, a molecular mass of approximately 4.5 Mb was calculated for the genome of E. amylovora.     

Serotyping of Vibrio anguillarum

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.     

Serotyping of Vibrio anguillarum.

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.     

西北地区甘草根瘤菌的表型多样性研究*

选用分离自西北干旱、半干旱地区的甘草根瘤菌68株和34株参比菌株,进行了113项表型性状测定,结果表明：不同地理来源、同一地理来源、甚至同一植株不同根瘤菌株在碳氮源利用、抗生素敏感性、抗逆性等方面存在着差异。部分菌株具有较强的耐盐耐碱能力,其中有5株和1株菌分别能耐受50g／L、60g／L的NaCl2有43％的菌株能在初始DH12的YMA培养基上生长。从数值分类树状图谱发现,在85．5％的相似水平上供试菌株构成了3个表观群。群I有27株菌,除4株来自陕西外,其余均来自新疆。群Ⅱ有4株菌,皆来自陕西。群Ⅲ有8株菌,6株来自陕西,2株来自宁夏,它们与R．hainanense聚在一起。而群I、群Ⅱ没有与参比菌株聚在一起,可能是新的表观群,其分类地位需进一步确定。     

Characterization of highly virulent Escherichia coli strains by ribosomal DNA restriction fragment length polymorphism

Patterns of ribosomal DNA polymorphism were examined to compare carboxylesterase B type B1 strains and B2 strains of Escherichia coli isolated from extra-intestinal infections. DNA from 14 type B2 strains showing the presence of alpha-haemolysin and mannose-resistant haemagglutinin and lethality to mice and 14 type B1 strains lacking these characteristics, was digested with HindIII, EcoRI, BamHI or BglII restriction enzymes and analysed by Southern blotting. The obtained ribotypes clearly differentiated the B2 strains from the B1 strains. These results indicate that genotypes of the highly virulent B2 strains are different from that of the less virulent B1 strains.     

Study of Vibrio anguillarum strains from different sources with emphasis on ecological and pathobiological properties.

A total of 317 Vibrio anguillarum strains were isolated from water, sediment, and diseased as well as healthy rainbow trout at a Danish mariculture farm and from feral fish caught close to the farm. All strains were examined serologically. Ten sera permitted determination of the O group in 66.7% of the strains from diseased rainbow trout. Furthermore, the O group could be determined in 45.1 to 65.4% of the strains from mucus, gills, and intestinal contents of healthy rainbow trout, while only 22.2 to 28.8% of the isolates from water, sediment, and gills or mucus of feral fish were groupable. Serogroup O1 and to some extent O2 appeared to be associated with trout. Strains from these serogroups were selected for analyses of hemagglutinating activity and surface hydrophobicity. Serogroup O1 comprised hemagglutinating as well as nonhemagglutinating strains; from cases of vibriosis, all O1 strains were nonhemagglutinating. The strains belonging to serogroup O2 were generally hemagglutinating. Examinations of surface hydrophobicity by salt aggregation and hydrophobic interaction chromatography suggested that the O1 strains were more hydrophobic than the O2 strains. In pathogenicity tests, O1 strains isolated from gills and mucus of healthy rainbow trout killed all trout in the test groups. A strain from the intestinal contents of healthy rainbow trout did not produce significant mortality. This strain could, however, be frequently reisolated from the pronephros of fish in the test group concerned. After challenge with strains from eel mucus and seawater, mortality was not produced, and furthermore, these strains could not be reisolated from the pronephros.     

Pulsed-field gel electrophoresis analysis of Vibrio vulnificus strains isolated from Taiwan and the United States

Vibrio vulnificus is a marine bacterium that causes human wound infections and septicemia with a high mortality rate. V. vulnificus strains from different clinical and environmental sources or geographic regions have been successfully characterized by ribotyping and several other methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminative method, but previous studies suggested that it was not suitable for examining the correlation of V. vulnificus strains from different origins. We employed PFGE to determine its efficacy for characterizing V. vulnificus strains from different geographic regions, characterizing a total of 153 strains from clinical and environmental origins from the United States and Taiwan after SfiI or NotI digestion. V. vulnificus strains showed a high intraspecific diversity by PFGE after SfiI or NotI digestion, and about 12% of the strains could not be typed by the use of either of these enzymes. For PFGE with SfiI digestion, most of the clinical and environmental strains from the United States were grouped into cluster A, while the strains from Taiwan were grouped into other clusters. Clinical strains from the United States showed a higher level of genetic homogeneity than clinical strains from Taiwan, and environmental strains from both regions showed a similarly high level of heterogeneity. PFGE with NotI digestion was useful for studying the correlation of clinical strains from the United States and Taiwan, but it was not suitable for analyzing environmental strains. The results showed that PFGE with SfiI digestion may be used to characterize V. vulnificus strains from distant geographic regions, with NotI being a recommended alternative enzyme.     

迟缓爱德华菌的外膜蛋白图谱及耐药性分析

分析 2 7株不同来源的迟缓爱德华菌 (Et)的外膜蛋白 (OMP) ,可分为A～H 8个型。 2 1株致病株与 6株非致病株有明显不同的图谱。致病株OMP条带多而深浓 ,并且相同来源的菌株有几乎一致的图谱。其中国内致病株以E型为主 ,与ATCC参考株相似。非致病株OMP条带则浅而稀疏 ,来源虽不同 ,但图谱极类似。另外 ,6株非致病株对磺胺、庆大、四环素等抗生素普遍耐药 ,而致病株除少数几株外 ,均对抗生素有不同程度的敏感     

Plasmids in Listeria monocytogenes in relation to cadmium resistance.

One hundred and seventy-three unrelated Listeria monocytogenes strains isolated from humans, animals, the environment, and food were analyzed for the presence of plasmids. Extrachromosomal DNA was found in 28% of the strains. Plasmid DNA was extracted more frequently from L. monocytogenes serogroup 1 strains (35%) than from serogroup 4 strains (15%). Among strains from food and the environment, 40% and 29%, respectively, harbored plasmids, whereas only 13% of the strains from humans and animals with listeriosis bore plasmids. We also investigated the susceptibility of 90 strains to seven antibiotics and four heavy-metal salts. No antibiotic resistance could be detected, but 95.3% of the plasmid-positive strains and only 12.7% of the plasmid-negative strains were resistant to cadmium. The plasmid-determined genetic basis of cadmium resistance was proven by conjugation between strains of L. monocytogenes and by cure of the plasmid. This is the first time that plasmids of L. monocytogenes have been shown to be associated with cadmium resistance.     

Characteristics of poliovirus strains isolated in Poland during 1981-1989 using monoclonal antibodies and determination of nucleotide sequence in viral RNA]

In this study currently used methods for analysis of poliovirus with monoclonal antibodies and determination of nucleotide sequence in viral RNA are presented. Twenty one strains of polioviruses isolated in Poland between 1981 and 1989 were tested. Nineteen of these strains were determined as to be derived from attenuated strains, including 10 strains isolated from 9 poliomyelitis patients. Two strains, one of type 1 isolated in 1984 and one of type 2 isolated in 1982 from children with meningitis were determined to be wild strains. The results obtained with monoclonal antibodies were confirmed by partial nucleotide sequence by dideoxy primer extension method. Analysis of poliovirus strains circulating in Poland in last 15 years suggest that the elimination of wild strains was achieved and sporadic isolation of wild strains is due most probably to imported strains.     

The effect of Aeromonas strains on the growth of Legionella

Thirty-one environmental and two type strains of Aeromonas were tested for their ability to inhibit the growth of strains of several Legionella species. Of 10 Legionella spp. tested, only Legionella pneumophila and Leg. longbeachae were able to resist the inhibitory effects of some of the Aeromonas strains. Selected Aeromonas strains were also tested for their ability to inhibit the growth of several non-legionella strains. None of the non-legionella strains were inhibited by any of the selected Aeromonas strains. Attempts were made to isolate Aeromonas strains from cooling towers and Aeromonas hydrophila was isolated from one of the cooling towers tested. The results suggest that many strains of Aeromonas can inhibit the growth of Legionella strains on solid media and could affect the isolation of legionellas from water sources.     

The interchangeability of siderophores in the Enterococcus genus

The functional interchangeability of siderophores was tested among 62 strais belonging to 12 species of genus Enterococcus. Most investigated strains were from E. faecalis and E. faecium species. The majority of examined enterococcal strains appeared highly resistant to EDDA (ethylene-di-amine-di-ortho-hydroxyphenylacetic acid), therefore the group of sensitive strains involved only 11 used as indicator strains. The determination of interchangeability of siderophores within enterococcal strains was performed using EDDA-agar media into which the indicator strains were included. Test colonies (donor strains) were applied to the surface of the media to determine whether the indicator organisms could obtain the required iron for growth by utilizing chelators from the test colony. Only two strains: E. solitarius DSM 5634 and E. pseudoavium DSM 5632 did not demonstrate the ability to utilize siderophores synthesized by all investigated strains. The other tested indicator strains appeared to be recipients of siderophores from 20-52 donor enterococcal strains. The ability to exchange siderophores in enterococci was found as the feature characterizing individual strains.     

Study of Vibrio anguillarum strains from different sources with emphasis on ecological and pathobiological properties

A total of 317 Vibrio anguillarum strains were isolated from water, sediment, and diseased as well as healthy rainbow trout at a Danish mariculture farm and from feral fish caught close to the farm. All strains were examined serologically. Ten sera permitted determination of the O group in 66.7% of the strains from diseased rainbow trout. Furthermore, the O group could be determined in 45.1 to 65.4% of the strains from mucus, gills, and intestinal contents of healthy rainbow trout, while only 22.2 to 28.8% of the isolates from water, sediment, and gills or mucus of feral fish were groupable. Serogroup O1 and to some extent O2 appeared to be associated with trout. Strains from these serogroups were selected for analyses of hemagglutinating activity and surface hydrophobicity. Serogroup O1 comprised hemagglutinating as well as nonhemagglutinating strains; from cases of vibriosis, all O1 strains were nonhemagglutinating. The strains belonging to serogroup O2 were generally hemagglutinating. Examinations of surface hydrophobicity by salt aggregation and hydrophobic interaction chromatography suggested that the O1 strains were more hydrophobic than the O2 strains. In pathogenicity tests, O1 strains isolated from gills and mucus of healthy rainbow trout killed all trout in the test groups. A strain from the intestinal contents of healthy rainbow trout did not produce significant mortality. This strain could, however, be frequently reisolated from the pronephros of fish in the test group concerned. After challenge with strains from eel mucus and seawater, mortality was not produced, and furthermore, these strains could not be reisolated from the pronephros.     

Plasmids in Listeria monocytogenes in relation to cadmium resistance.

One hundred and seventy-three unrelated Listeria monocytogenes strains isolated from humans, animals, the environment, and food were analyzed for the presence of plasmids. Extrachromosomal DNA was found in 28% of the strains. Plasmid DNA was extracted more frequently from L. monocytogenes serogroup 1 strains (35%) than from serogroup 4 strains (15%). Among strains from food and the environment, 40% and 29%, respectively, harbored plasmids, whereas only 13% of the strains from humans and animals with listeriosis bore plasmids. We also investigated the susceptibility of 90 strains to seven antibiotics and four heavy-metal salts. No antibiotic resistance could be detected, but 95.3% of the plasmid-positive strains and only 12.7% of the plasmid-negative strains were resistant to cadmium. The plasmid-determined genetic basis of cadmium resistance was proven by conjugation between strains of L. monocytogenes and by cure of the plasmid. This is the first time that plasmids of L. monocytogenes have been shown to be associated with cadmium resistance.